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Biotechnology Consultation
Note to the File
BNF No. 000093
Date: June 28, 2004
Subject: Mycogen Seeds c/o Dow AgroSciences LLC (DAS) BNF 000093 (B.t. Cry1F maize line 6275)
Keywords: Cry1F, PAT, phosphinothricin acetyltransferase, maize, corn, European corn borer, glufosinate
In a submission to FDA dated June 30, 2003, DAS provided data and information to support their safety and nutritional assessment of their insect resistant, glufosinate tolerant maize (corn) line containing transformation event TC6275 (hereinafter maize line 6275). DAS provided additional information dated September 5, November 10, and December 4, 2003.
DAS states that maize line 6275 was derived from public inbred line Hi-II by transformation with DNA containing the maize-optimized, truncated cry1F (mocry1F) gene derived from the Bacillus thuringiensis (B.t.) var. aizawai strain PS811, the bar gene from Streptomyces hygroscopicus, and the regulatory sequences necessary for expression of both genes.1 The mocry1F gene encodes a truncated version of the Cry1F protein which provides a means of controlling the European corn borer and certain other Lepidopterous pests of maize. The bar gene encodes the enzyme phosphinothricin acetyltransferase (PAT) which confers tolerance to glufosinate-ammonium herbicides and acts as a selectable marker.2
The Environmental Protection Agency (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 Cry1F protein in maize line 6275 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.
Maize line 6275 was created by Agrobacterium-mediated transformation of public inbred line Hi-II with the binary T-DNA vector PHP 12537 which carries the mocry1F and bar transgenes. The T-DNA region of PHP 12537 contains the following elements intended to be transferred to the recipient plant line.
| Name | Description |
|---|---|
| RB | right border |
| UBIZM1(2) | ubiquitin promoter (plus intron and 5' untranslated sequence) from Zea mays |
| mocry1F (trunc) | synthetic, maize-optimized version of truncated Cry1F from Bacillus thuringiensis var. aizawai |
| PINII | terminator sequence from Solanum tuberosum proteinase inhibitor II |
| CaMV 35S-1841 enhancer | upstream enhancer from Cauliflower Mosaic Virus strain 1841 |
| CaMV 35S-1841 promoter | 35S promoter from Cauliflower Mosaic Virus strain 1841 |
| ADH1 | alcohol dehydrogenase intron 1 from Zea mays |
| bar | phosphinothricin acetyltransferase gene isolated from Streptomyces hygroscopicus |
| PINII | terminator sequence from Solanum tuberosum proteinase inhibitor II |
| LB | left border |
Transformation vector PHP 12537 contains the spc and tet genes which encode resistance to the antibiotics spectinomycin and tetracycline, respectively. The spc and tet genes are located outside the T-DNA border sequences of the vector; therefore, the genes are not expected to be transferred to the recipient plant line during transformation.
DAS states that Southern hybridization was used to determine the nature and number of mocry1F and bar gene insertions in maize line 6275. This analysis revealed that there is a single T-DNA insert in maize line 6275 and that the bar gene transcription unit was inserted intact but the mocry1F gene transcription unit containing the maize ubiquitin promoter was truncated at the 5' end. DAS also states that Southern hybridization analysis revealed that, as expected, maize line 6275 does not contain either the spectinomycin or tetracycline resistance genes.
DAS states that Southern hybridization was used to assess the genetic stability of the insert in three separate generations of maize line 6275. This analysis revealed that the insert in maize line 6275 was stable across the three separate generations examined. DAS also states that data from segregation analysis indicate that the insertion in maize line 6275 is a stable insertion and that it shows a Mendelian inheritance pattern.
DAS states that maize has traditionally been used as human food (meal, flour, oil, high fructose syrup, etc.) and animal feed (grain, silage, gluten feed). Maize line 6275 will be grown using the same commercial maize cultivation practices that are currently used by maize growers, and it will be used for the same commercial uses as currently commercialized maize varieties. DAS states that the primary use of maize is for animal feed, but maize is also processed into food and industrial products. DAS concludes that maize line 6275 is suitable for all current applications of commercial maize.
DAS analyzed grain and forage samples from field trials at six locations, with three replicates per location. DAS measured components of grain and forage from maize line 6275 and its near isogenic control line (CHPH09B/2MW; hereafter the control line) and compared the measured values from maize line 6275, the control line, and reported literature ranges.
DAS analyzed forage from maize line 6275 and the control line harvested at the kernel dough stage (R4) for:
DAS reports that the mean level of fat in maize line 6275 was less than that in the control line (p<0.05). DAS also reports that the mean levels of ash, carbohydrates, and calcium in maize line 6275 were greater than those in the control line (p<0.05). DAS reports that mean levels for all analyzed components of forage from maize line 6275 are within the published literature range.
DAS analyzed grain harvested at physiological maturity (R6, 15% moisture) from maize line 6275 and the control line for:
DAS reports that the mean levels of crude fiber, ADF, linoleic acid, lysine, calcium, zinc, vitamin B1, and folic acid in maize line 6275 were less than those in the control line (p<0.05). DAS also reports that the mean levels of oleic acid, isoleucine, arginine, glutamic acid, proline, iron, vitamin A, and vitamin E in maize line 6275 were greater than that in the control line (p<0.05). DAS reports that the levels of vitamin B2 in maize line 6275 and the near isogenic control line are below the quantitative limits for the analytical method used. DAS reports that mean levels for all measurable components of grain from maize line 6275 are within the reported literature range except for vitamin A.
DAS reports data from a study in which vitamin A levels4 were measured in maize line 6275, maize line 6275 sprayed with glufosinate-ammonium herbicide, and the control line. DAS reports that mean vitamin A levels in maize line 6275 sprayed with glufosinate-ammonium herbicide are not different from the control line and are within the reported literature range. DAS states that the mean vitamin A level in maize line 6275 is greater than that for the control line and greater than the reported literature range (p<0.05). DAS concludes that differences in measured vitamin A values and those reported in the literature are not due to the presence of the transgene but are more likely from differences in the assay procedures that were used.
DAS states that maize line 6275 is as safe as the control line and other maize lines used for food and feed products. DAS states that the range of all proximates (including ADF and NDF), amino acids, fatty acids, minerals, vitamins (with the exception of vitamin A), secondary metabolites, and anti-nutrients are within published literature range values.
DAS has concluded that their insect resistant, glufosinate tolerant maize line 6275 is not materially different in safety, composition, or any other relevant parameter from maize now grown, marketed, and consumed. At this time, based on DAS's data and information, the Agency considers DAS's consultation on maize line 6275 to be complete.
/s/
Jason Dietz
(1)DAS describes maize-optimization as altering the G+C content of the native bacterial gene to more closely approximate the higher G+C content typically found in maize genes.
(2)In its submission, DAS refers to the phosphinothricin acetyltransferase protein encoded by the bar gene as BAR to distinguish it from the phosphinothricin acetyltransferase protein encoded by the pat gene. To be consistent with FDA's previous notes to the file and response letters regarding crops containing the phosphinothricin acetyltransferase protein encoded by the bar gene, FDA refers to the phosphinothricin acetyltransferase protein from the bar gene as PAT in the note to the file and response letter for BNF 000093.
(3)Moisture content on each of the samples was determined so that the data could be expressed on a dry matter basis.
(4)When maize is used as animal feed, it is normally supplemented with vitamin A because the level of vitamin A in maize is low compared to the requirements of domestic animals (Vitamin Tolerance of Animals. 1987. National Academy of Science pp. 3-10).
