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Memorandum to the File for FMF No. 000763

Food Master Files & Food Additive Petitions Main Page

CFSAN/Office of Food Additive Safety
August 29, 2006

Memorandum to the File
FMF No. 000763

Date: August 29, 2006

Subject: Syngenta Seeds, Inc.'s submission regarding Bt 10 corn

Keywords: corn, Bt 10, Syngenta, Cry1Ab, PAT, Food Master File Number 763


In a submission dated June 8, 2005, Syngenta Seeds, Inc. (Syngenta) submitted to the Food and Drug Administration (FDA, the agency) a safety and nutritional assessment of grain derived from genetically engineered corn event Bt 10 (Bt 10 corn). Syngenta submitted additional information on July 1, July 19, July 26, August 1, August 30, September 1, September 23, September 29, December 12, 2005 and January 24, January 30, February 8, and February 14, 2006. Syngenta explained that event Bt 10 was introduced into several corn lines when event Bt 10 was mistakenly thought to represent corn event Bt 11, the subject of a previously completed consultation with FDA (BNF 000017). Syngenta determined that due to its mistaken identification of the Bt 10 corn lines as Bt 11 corn lines, approximately 37,000 acres in the United States were planted with Bt 10 corn, and most of the harvested grain went into the food and feed supply. Although Syngenta is no longer marketing the misidentified Bt 10 lines, and notes that it is not pursuing commercial approval for Bt 10 corn in the United States, Syngenta provided FDA with these submissions to address issues ordinarily addressed in a pre-market voluntary consultation for corn grain.


Syngenta states that in December 2004, the firm determined that it had inadvertently marketed Bt 10 corn. Syngenta notified FDA of the inadvertent marketing of Bt 10 corn in a meeting with the agency on January 27, 2005. In a letter to FDA dated February 7, 2005, Syngenta states that the Cry1Ab and PAT proteins produced in Bt 10 corn fall within the existing tolerance exemptions for these proteins established by the Environmental Protection Agency (EPA) under Section 408 of the Federal Food, Drug, and Cosmetic Act. In its letter, Syngenta also discusses the fact that there are no significant native toxins or allergens in corn.

On April 27, 2005, FDA issued a statement on Bt 10 corn in which it said," FDA does not believe that possible unintended changes in the composition of corn pose food or feed safety risks or regulatory issues in circumstances in which the corn makes up a small part of the total food or feed supply. In this type of situation, the relevant information for food and feed safety is the safety of the new protein(s) in the corn. Therefore, in circumstances such as those surrounding the presence of Bt 10 corn in food and feed, the information relevant to safety assessment is limited to the safety of the proteins evaluated by EPA. Based on EPA's finding that the genetically engineered proteins in Bt 10 co rn are safe, the extremely low levels of Bt 10 corn in the food and feed supply, and the fact that corn does not contain any significant natural toxins or allergens, FDA has concluded that the presence of Bt 10 corn in the food and feed supply poses no safety concerns." (The statement is available at http://www.fda.gov under the topic Food on the Announcements page of the Biotechnology section.)

Intended Effect:

Bt 10 corn is genetically engineered to express the Cry1Ab protein and the phosphinothricin acetyltransferase (PAT) protein. The Cry1Ab protein provides resistance to certain lepidopteran insect pests. The PAT protein provides tolerance to glufosinate ammonium herbicide.

Regulatory considerations:

EPA regulates plant incorporated protectants under the Federal Food, Drug, and Cosmetic Act and the Federal Insecticide, Fungicide, and Rodenticide Act. Under EPA regulations, the Cry1Ab protein in Bt 10 corn is considered a pesticidal substance and the PAT protein is considered an inert ingredient. Therefore, the safety assessment of these proteins falls under the regulatory purview of EPA.

Development of Bt 10 corn:

Syngenta states that Bt 10 corn was developed by transformation using the pZO1502 transformation vector and a polyethylene glycol-mediated protoplast transformation/regeneration system. Transformants were selected using synthetic media containing glufosinate.

Components of transformation vector pZO1502
Genetic element Description
35S promoter Promoter derived from Cauliflower Mosaic Virus. Two copies of this promoter are contained in pZO1502, one to promote expression of the cry1Ab gene and one to promote expression of the pat gene.
IVS2 and IVS6 introns These introns were derived from the corn alcohol dehydrogenase 1S gene. These sequences are used to enhance expression of the pat and cry1Ab genes, respectively.
nos terminator The nos terminator was derived from the nopaline synthase gene from Agrobacterium tumefaciens.
cry1Ab A modified version of the full-length cry1Ab gene originally derived from Bacillus thuringiensis var. kurstaki strain HD-1.
pat A modified version of the phosphinothricin acetyltransferase gene derived from the soil microorganism Streptomyces viridochromogenes strain Tu494.
bla1 A beta-lactamase gene under the regulatory control of a prokaryotic promoter. Expression of this gene in bacterial cells confers tolerance to and allows cell division in the presence of certain beta-lactam antibiotics.
ColE1-ori An origin of replication that permits replication of transformation vector pZO1502 in bacterial cells. No protein is encoded by this sequence.

1In its submissions, Syngenta also refers to the bla gene as amp.

Genetic modification and molecular characterization:

Using Southern analyses, genomic cloning, and DNA sequencing of cloned fragments of the insert and polymerase chain reaction (PCR) products, Syngenta was able to characterize the insertion in the Bt 10 event. Within the insertion is a repetition of the pZO1502 starting plasmid that creates multiple copies of full length cry1Ab and pat genes. The repeated genes contain their respective regulatory sequences that allow expression in corn. Syngenta confirmed that the full length cry1Ab and pat genes are identical to the genes found in Bt 11 and in the pZO1502 starting plasmid.

The complex insertion event in the plant contains a number of bacterial elements that were used in the construction of the transformation vector. These include the bla gene and multiple copies of the ColE1 origin of replication (ori). While these bacterial elements do not function in plants, Syngenta believes that the presence of multiple copies of ColEI-ori in the Bt 10 insertion event prevented them from cloning the full length insert in their bacterial system because the multiple origins of replication made the clones unstable. However, Syngenta was able to characterize most of the junctions and elements internal to the insertion event by using a strategy to generate libraries with clones containing shorter portions of the insert without a complete ColE1 element.

From a number of different clones, Syngenta showed that a portion of the insert in Bt 10 corn contains a number of short, truncated and scrambled vector elements. Neither open reading frames (ORFs) likely to be expressed in this region, nor appropriate regulatory elements for expression were found to be present. Syngenta confirmed the presence of this scrambled portion of the Bt 10 insert by PCR analyses of genomic DNA.

Syngenta identified and sequenced the right and left border junctions of the Bt 10 insertion event. Corn DNA at the right junction matches sequence that has previously been reported to be derived from Bin 1.07 chromosomal location. This corroborates earlier mapping of the Bt 10 insertion. Syngenta also showed that the Bt 10 insertion event is contained on a single Swa I restriction fragment of approximately 27 kilobase pairs in length. Syngenta performed an ORF analysis of the sequences found at these junctions and stated that they did not identify any new ORFs greater than 50 amino acids with the regulatory elements necessary for expression.

The complex nature of the insertion event with multiple repeat and truncated, scrambled elements precluded Syngenta from characterizing the complete linear relationship of the various clones. Syngenta stated that several different genomic libraries of Bt 10 corn DNA yielded identical clones multiple times, and analysis of those clones did not show any ORFs with the necessary promoter elements to be expressed (aside from the expected Cry1Ab, and PAT) or for read-through from corn DNA into the insert DNA to occur. Syngenta concluded, based on data from Western blot analyses for proteins encoded by the bla (not expressed), cry1Ab, and pat genes, Southern analyses, and DNA sequencing of clones representative of the insert, that there are no unintended proteins expressed in Event Bt 10.

Mapping and co-segregation analysis:

Syngenta analyzed the number of insertion loci of the cry1Ab (35S promoter/IVS6 intron/cry1Ab/nos terminator) and pat (35S promoter/IVS2 intron/pat/nos terminator) gene expression cassettes in Bt 10 corn using mapping and co-segregation analysis of the Cry1Ab and PAT-expressing genes. These analyses were performed on a BC1 (back-cross 1) generation of seed progeny generated by crossing a hybrid line of Bt 10 corn (presumed hemizygous for both the cry1Ab and pat genes) with a non-transgenic line. Segregation analysis revealed that the glufosinate ammonium herbicide tolerance trait in Bt 10 corn segregated in a 1:1 ratio consistent with a single functional pat locus.

Syngenta confirmed the linkage between the pat and cry1Ab genes by testing glufosinate-tolerant plants for expression of the Cry1Ab protein using a lateral flow dipstick assay. In addition, Syngenta used polymerase chain reaction (PCR) analysis to confirm that glufosinate-tolerant plants also contained the cry1Ab gene. Syngenta concludes that the segregation data and the molecular data are consistent with a single locus of insertion of the functional pat and cry1Ab gene expression cassettes. Additionally, Syngenta used 60 molecular markers distributed throughout the corn genome to map the single locus of insertion of the functional pat and cry1Ab gene expression cassettes to chromosome 1, Bin 1.07 of the corn genome.

Compositional Analysis:

Syngenta analyzed key nutritional components in mature grain derived from two Bt 10-derived field corn hybrids using recognized analytical methods. One hybrid was grown at five geographical locations while the second hybrid was grown at four geographical locations. For each hybrid, Syngenta collected one sample from each location and analyzed it for the following components:

Proximates: ash, fat, moisture, protein, carbohydrate, acid detergent fiber (ADF), neutral detergent fiber (NDF).
Minerals: calcium, copper, iron, magnesium, manganese, phosphorus, potassium, zinc.
Amino acids: aspartic acid, threonine, serine, glutamic acid, proline, glycine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, alanine, cysteine, valine, lysine, arginine, and tryptophan.
Fatty acids: palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), and linolenic acid (18:3).
Vitamins: folic acid, vitamin B1, vitamin B2, vitamin E (total).
Secondary metabolites and anti-nutrients: inositol, phytic acid, raffinose, ferulic acid, p-coumaric acid, and trypsin inhibitor.

Syngenta compared the mean level of each component to the ranges of levels reported for corn grain in the literature and publicly available databases. Syngenta noted that the levels of all components evaluated in their study were within the ranges reported for corn grain.


FDA finds that the data and information in Syngenta's submissions are consistent with and provide additional support for FDA's April 27, 2005, statement that the presence of Bt 10 corn grain in the food and feed supply poses no safety concerns, and are consistent with and provide support for Syngenta's conclusion that grain from Bt 10 corn is not materially different in composition, safety or other relevant parameters from corn grain now grown, marketed, and consumed.


Mary D. Ditto, Ph.D.


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