Biotechnology Consultation Note to the File BNF No. 000113
Return to inventory: Submissions on Bioengineered New Plant Varieties
Date: December 1, 2008
Subject: Biotechnology Notification File (BNF) 113, Bacillus thuringiensis Vip3Aa20 and Escherichia coli phosphomannose isomerase (PMI) proteins; maize event MIR162
Keywords: Maize, corn, Zea mays L., MIR162, lepidopteran resistance, Vip3Aa20, vip3Aa20 gene, Bacillus thuringiensis, phosphomannose isomerase (PMI), pmi gene (or manA gene), Escherichia coli
In a submission dated August 3, 2007, Syngenta Seeds, Inc. (Syngenta), submitted to FDA a safety and nutritional assessment of genetically engineered lepidopteran resistant maize, transformation event MIR162 (hereafter referred to as maize event MIR162). Syngenta provided additional information on December 17, 2007, and March 31, 2008. Syngenta concluded that food and feed derived from maize event MIR162 are as safe and nutritious as food and feed derived from conventional maize.
2. Intended Effect
The intended effect of the modification in maize event MIR162 is to confer resistance to lepidopteran insect pests of maize. To accomplish this objective, Syngenta introduced the vip3Aa20 gene which encodes the Vip3Aa20 protein that confers resistance to several lepidopteran insect pests, including corn earworm, black cutworm, fall armyworm, and western bean cutworm. The vip3Aa20 gene is a variant of the native insecticidal vip3Aa19 gene derived from Bacillus thuringiensis (B. thuringiensis).
Syngenta also introduced the phosphomannose isomerase (pmi) gene from Escherichia coli (E. coli) which encodes the PMI protein for use as a selectable marker during the development of maize event MIR162. Nontransgenic maize cells cannot grow on media containing mannose as the sole or primary energy source; however, transgenic maize cells expressing PMI protein are able to grow on such media.
3. Regulatory Considerations
The Environmental Protection Agency (EPA) defines a plant-incorporated protectant (PIP) as "a pesticidal substance that is intended to be produced and used in a living plant, or the produce thereof, and the genetic material necessary for the production of such a pesticidal substance," including "any inert ingredient contained in the plant, or produce thereof" (40 CFR 174.3). EPA regulates PIPs under the Federal Food, Drug, and Cosmetic Act and the Federal Insecticide, Fungicide, and Rodenticide Act. Under EPA regulations, the vip3Aa20 gene in maize event MIR162 and resulting expression products are considered pesticidal substances; and the pmi gene and resulting expression products are considered inert ingredients. EPA considers the recombinant DNA construct containing the vip3Aa20 and pmi genes to be part of the PIP in maize event MIR162, and therefore EPA is reviewing the recombinant DNA construct and resulting expression products.
4. Genetic Modifications and Characterization
4.1 Transformation Plasmid and Method
To generate maize event MIR162, Syngenta used an Agrobacterium tumefaciens (A. tumefaciens)-mediated transformation system. Syngenta transformed immature maize embryos of variety NP2500 x NP2499 using the pNOV1300 plasmid. This plasmid contains two expression cassettes within the transfer-DNA (T-DNA) region: the vip3Aa19 gene cassette and the pmi gene cassette. The E. coli spectinomycin (spec or aadA) antibiotic resistance marker gene is located outside of the T-DNA region. The details of the two cassettes within the T-DNA region are:
- The vip3Aa19 gene cassette contains the coding sequence of the vip3Aa19 gene under the control of two regulatory elements, the ZmUbiInt promoter region from the Zea mays (Z. mays) polyubiquitin gene and the 35S terminator from the cauliflower mosaic virus 35S RNA.
- The pmi gene cassette contains the coding sequence of the pmi gene (also known as manA) from E. coli under the control of the ZmUbiInt promoter region from the Z. mays polyubiquitin gene and the NOS terminator from the A. tumefaciens nopaline synthase gene polyadenylation sequence.
Following transformation of the immature maize embryos, Syngenta selected maize event MIR162 for further development based on the presence of the pmi and vip3Aa genes, the absence of the spec gene, agronomic performance, and resistance to insect feeding damage.
4.2 Characterization, Inheritance, and Stability of the Introduced DNA
In its submission, Syngenta noted that the vip3Aa19 gene from the pNOV1300 plasmid underwent a transformation-induced sequence change, as detected by DNA sequence analysis of the insert, and was subsequently designated by Syngenta as the vip3Aa20 gene. The Vip3Aa20 protein contains a single amino acid change (methionine to isoleucine at position 129) when compared to the Vip3Aa19 protein. For clarity in this Note-to-File, FDA refers to the resulting gene cassette as the vip3Aa20 gene cassette, the gene as the vip3Aa20 gene, and its encoded protein as the Vip3Aa20 protein.
Syngenta characterized the insert in maize event MIR162 using restriction enzyme digestion of genomic DNA followed by Southern blot analyses. In its submission, Syngenta stated that data from Southern analyses demonstrated that maize event MIR162 contains a single, intact insert containing one copy of both the vip3Aa20 and pmi gene cassettes. Syngenta confirmed that no backbone sequences (i.e., DNA from outside of the T-DNA region) from transformation plasmid pNOV1300 are present in maize event MIR162.
Syngenta studied the inheritance of the vip3Aa20 and pmi gene traits in three generations (BC1F1, BC2F1, and BC4F1) of maize event MIR162. Based on the results of Chi square analysis of trait inheritance data, Syngenta concluded that the vip3Aa20 and pmi genes are inherited in a predictable manner according to Mendelian principles and that this inheritance pattern is consistent with the stable integration of the insert in maize event MIR162 at a single site in the maize genome.
Syngenta assessed the stability of the DNA insert in three generations (BC1F1, BC2F1, and BC4F1) of maize event MIR162 using restriction enzyme digestion of genomic DNA followed by Southern blot analysis. According to Syngenta, analysis of these three generations of maize event MIR162 yielded the expected hybridization pattern – a single band of a consistent size. On this basis, Syngenta concluded that the insert in maize event MIR162 is stable during conventional breeding over multiple generations.
5. Food and Feed Use
Syngenta states that maize event MIR162 will be grown for the same uses as maize varieties currently commercially available in the United States. While maize grown in the United States is predominantly (roughly 60 percent) used to feed domestic animals, either as grain or silage, maize grain is also processed by wet or dry milling to yield food products such as high fructose corn syrup, starch, oil, grits, and flour. Non-food/feed purposes for grain include use for fuel ethanol production; however, the by-products of such industrial distilling processes may also be used in animal feeds. In its submission, Syngenta states that maize event MIR162 is suitable for the same uses as conventional maize.
6. Compositional Analysis
To confirm that food and feed derived from maize event MIR162 are nutritionally comparable to and as safe as food and feed derived from conventional maize, Syngenta compared the composition of forage and grain from a transgenic maize event MIR162 hybrid to a nontransgenic, near-isogenic hybrid. For the compositional analysis, Syngenta used a transgenic maize hybrid of the genotype NP2276(MIR162)/NP2391 (hereafter referred to as the MIR162 hybrid) and a nontransgenic, near-isogenic maize hybrid of the genotype NP2276/NP2391 (hereafter referred to as the near-isogenic hybrid). Syngenta analyzed forage for levels of acid detergent fiber (ADF), neutral detergent fiber (NDF), proximates, and minerals. Syngenta analyzed grain for levels of ADF, NDF, proximates, minerals, vitamins, amino acids, fatty acids, secondary metabolites and anti-nutrients. The specific analytes measured in forage and/or grain are listed in Table 1.
|Fiber and Proximates||Minerals||Vitamins||Amino Acids||Fatty Acids||Secondary Metabolites||Anti-Nutrients|
acid detergent fiber (ADF)*
neutral detergent fiber (NDF)*
total dietary fiber (TDF)
Folic Acid (B9)
|The asterisk denotes analytes measured in both forage and grain. All other listed analytes were measured in grain only.|
6.1 Testing Strategy
Syngenta analyzed the composition of forage and grain tissues collected from MIR162 hybrid and near-isogenic hybrid maize grown in six field locations in the United States during 2005. At each location, plants were grown using a randomized complete block design, with three replicates for each genotype, yielding a total of 18 samples each for forage and grain compositional analyses (N=18). The plants were self-pollinated by hand and the developing ears were bagged to avoid cross-pollination. For the forage samples, the entire above-ground portion of the plants was harvested at dough stage (R4), the stage at which silage would typically be prepared. For the grain samples, grain was shelled from ears harvested after physiological maturity (R6).
In its submission, Syngenta provided the average and the range for each analyte included in the compositional analysis of the MIR162 hybrid and near-isogenic hybrid.1 Syngenta conducted statistical analysis of the compositional data using variance across locations and determining the statistical significance of differences between the MIR162 hybrid and the near-isogenic hybrid using a standard F-test. Statistical significance was determined where the difference in the average level of a given analyte was greater than 5 percent (p-value<0.05).
In its submission, Syngenta provided published literature values2 for each analyte, where available,3 to assess whether statistically significant differences between the composition of the MIR162 hybrid and the near-isogenic hybrid maize are biologically meaningful.
6.2 Forage Analysis
Of the analytes measured in maize forage, the only statistically significant difference between the MIR162 hybrid and the near-isogenic hybrid was the average level of NDF. The average level of NDF in the MIR162 hybrid was statistically significantly higher than in the near-isogenic hybrid; however, it was within the published literature ranges. The average levels for all the other analytes also fell within the published literature ranges.
6.3 Grain Analysis
6.3.1 Fiber and Proximates
Statistically significant differences between the MIR162 hybrid and the near-isogenic hybrid were observed in the average levels of ash, NDF, and starch. The average levels of ash and NDF were statistically significantly higher and the average level of starch was statistically significantly lower in the MIR162 hybrid than in the near-isogenic hybrid. However, the average levels of ash, NDF, and starch in the MIR162 hybrid were within the published ranges.
The average levels of calcium, iron, and phosphorus were statistically significantly higher in the MIR162 hybrid than in the near-isogenic hybrid. However, the average levels of these minerals were within the published literature ranges as were the other minerals measured, with the exception of selenium and sodium. Some of the values for selenium and all of the values for sodium in both the MIR162 hybrid and the near-isogenic hybrid were below the limit of quantification. However, low levels of selenium and sodium are consistent with values reported in the published literature and variable levels of these minerals found in soil in corn growing areas of the United States.
The average levels of Vitamin A, Vitamin B6, and Vitamin E were statistically significantly lower in the MIR162 hybrid than in the near-isogenic hybrid. However, the average levels of all measured vitamins in the MIR162 hybrid were within the ranges reported for the near-isogenic hybrid and the ranges reported in the ILSI database.
6.3.4 Amino Acids
No statistically significant differences between the MIR162 hybrid and the near-isogenic hybrid were observed in the average levels of any of the eighteen amino acids measured. Further, the levels of all amino acids measured were within the published literature ranges.
6.3.5 Fatty Acids
The average level of linoleic acid was statistically significantly lower and the average level of linolenic acid was statistically significantly higher in the MIR162 hybrid than in the near-isogenic hybrid. However, the average levels of both linoleic acid and linolenic acid were within the ranges reported for the near-isogenic hybrid and the ranges reported in the ILSI database as were the remainder of the fatty acids measured.
6.3.6 Secondary Metabolites and Anti-nutrients
The average levels of ferulic and p-coumaric acids were statistically significantly higher in the MIR162 hybrid than in the near-isogenic hybrid. However, the average levels of ferulic acid, p-coumaric acid, and the remainder of the secondary metabolites and anti-nutrients were within the published literature ranges. Syngenta noted that all of the values obtained from analyses for furfural and most of the values for raffinose from both MIR162 and the near-isogenic controls were below the limit of quantification.
6.4 Conclusions from Forage and Grain Compositional Analyses
On the basis of the results of its analyses of maize forage and grain composition, Syngenta concluded that forage and grain derived from the MIR162 hybrid is comparable to forage and grain derived from the near-isogenic hybrid.
7. Wholesomeness Study
Syngenta also provided the results of a 44-day broiler chicken study in which chickens were fed diets prepared with (1) MIR162 hybrid grain, (2) near-isogenic hybrid grain, or (3) a commercially available maize grain. Starter, grower, and finisher diets were formulated with maize contents ranging from 50% to 64% by weight, depending on the type of diet. The study included mortality, feed consumption and growth assessments (e.g., overall mean bodyweight, overall carcass yield, and yield of various carcass portions). Syngenta stated that there were no statistically significant differences in any of the parameters measured among birds fed diets prepared with the MIR162 hybrid grain and those fed diets prepared with the near-isogenic hybrid grain or the commercially available grain.
Syngenta has concluded that its lepidopteran resistant maize variety, maize event MIR162, and the food and feed derived from it are not materially different in safety, composition, or any other relevant parameters from maize now grown, marketed, and consumed. At this time, based on Syngenta's data and information, the agency considers Syngenta's consultation on maize event MIR162 to be complete.
Carrie McMahon, Ph.D.
1A value represents the average of three replicate plots per location, with grain from fifteen plants pooled from each plot. Some values obtained from analyses for selenium and raffinose and all of the values obtained from analyses for sodium and furfural in grain from the MIR162 hybrid and the near-isogenic hybrid were below the limit of quantification (LOQ) for the analytical method used and were excluded from the statistical analysis. Exclusion of these analytes did not, however, affect Syngenta's conclusions. Syngenta stated that the values for all four analytes across all locations are consistent with published values (including <LOQ values).
2Syngenta cited data from the following sources: (1) ILSI (2006). International Life Sciences Institute Crop Composition Database Version 3.0. http://www.cropcomposition.org; and (2) OECD (2002). Consensus document on compositional considerations for new varieties of maize (Zea mays): key food and feed nutrients, anti-nutrients and secondary plant metabolites. Publication No. 6, 2002. ENV/JM/MONO (2002) 25.
3The OECD maize composition document does not provide ranges for carbohydrates in forage, or for starch or manganese in grain.