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Biotechnology Consultation
Note to the File
BNF No.
000086
Date: June 5, 2003
From: Consumer Safety Officer
Subject: Biotechnology Notification File (BNF) BNF 000086: Glufosinate-Tolerant Cotton Transformation Event LLCotton25
To: Administrative File, BNF 000086
Cotton, Gossypium hirsutum L., Coker 312 variety, transformation event LLCotton25, herbicide-tolerance, Glufosinate-ammonium (GA), L-phosphinothricin, phosphinothricin-N-acetyltransferase (PAT), bialaphos resistance (bar) gene, Streptomyces hygroscopicus, strain ATCC21705
In a submission dated August 30, 2002, Bayer CropScience USA LP (formerly known as Aventis CropScience USA LP) submitted to FDA a summary of the safety and nutritional assessment they have conducted on a new line of genetically engineered herbicide-tolerant cotton, transformation event LLCotton25. The company provided additional information in a submission dated January 8, 2003. Bayer CropScience (Bayer) concludes that transformation event LLCotton25 is as safe and nutritious as conventional cotton varieties currently being marketed. Aventis CropScience USA LP, had previously completed consultations with FDA on herbicide-tolerant lines of corn (T14, T25; BNF 000029) and rice (LLRiceE06, LLRiceE62; BNF 000063). In each of these previous consultations, the herbicide tolerance was to glufosinate-ammonium (GA). In this current submission, the formulation of GA is known as Liberty®.
The intended effect of this genetic modification of cotton is to confer tolerance to the herbicide GA. To accomplish this objective, Bayer introduced the bialaphos resistance (bar) gene from Streptomyces hygroscopicus, strain ATCC21705 into the cotton variety Coker 312. The bar gene encodes the enzyme phosphinothricin-N-acetyltransferase (PAT). This enzyme catalyzes the conversion of L-phosphinothricin, the active ingredient in GA, to an inactive form. Plants expressing the bar gene product PAT, can inactivate the herbicide GA and as a result are herbicide-tolerant.
Bayer states that the company submitted a petition to USDA for non-regulated status of transformation event LLCotton25. On March 11, 2003, Bayer informed FDA by telephone that the USDA petition was finalized and LLCotton25 was de-regulated. Bayer applied to EPA for registration of the use of Liberty® in conjunction with transformation event LLCotton25.
Cotton, Gossypium hirsutum L., is grown world-wide, primarily as a source of fiber in the textile manufacturing industry. Cottonseed is a by-product of fiber production. Cottonseed contains natural toxicants, gossypol, and cyclopropenoid fatty acids. Cottonseed is processed into four major products: oil, meal, hulls, and linters. Cottonseed oil and to a lesser extent processed linters are routinely used in human food and have a long history of safe use. Cottonseed, cottonseed meal and hulls, are used in animal feed.
Cottonseed oil intended for human consumption is highly purified. The purification process substantially reduces the content of cyclopropenoid fatty acids and gossypol. The refined cottonseed oil is used for frying and cooking, and in various foods including mayonnaise, salad dressing, shortening, and margarine. Linters are also highly processed to obtain pure cellulose for use in food, for example, in casings for bologna, sausages, and frankfurters, and in products such as ice cream and salad dressings.
Whole cottonseed is used as a feed, supplying energy and fiber for dairy cows. Cottonseed meal is used as a protein supplement in animal feed. Cottonseed meal is processed to reduce the content of gossypol and cyclopropenoid fatty acids to acceptable levels. The hull is a protective coating of the seed removed prior to processing cottonseed into oil and meal. Hulls are used as a high fiber component of livestock feeds due to their high cellulose and lignin content.
Bayer used Coker 312 as the parental variety for transformation. Coker 312 is a United States Protected Variety of SEEDCO Corporation (PVP 7200100). Coker 312 was developed from a cross of Coker 100 with (Delta and Pine Land) D&PL-15 and selected through successive generations of line selection.
Plasmid vector pGSV71 was maintained in Escherichia coli, and transferred to a suitable Agrobacterium tumefaciens strain prior to plant transformation. The transfer of the plasmid vector pGSV71 into the cotton genome was thus Agrobacterium-mediated and resulted in transfer of the DNA only between the T-DNA border repeats. The resulting chimeric gene is designated as P35S-bar-3'nos and contains a single open-reading frame. The transformation was performed by exposing tissue excised from between the hypocotyl and the radicle of three-days post-emergent cotton seedlings to a culture of A. tumefaciens harboring the Ti plasmid pGV3000 and the plasmid vector pGSV71. After co-culture, the explants were regenerated to whole plants using the appropriate regeneration media with 500 mg/L claforan to eliminate residual A. tumefaciens. Transformed plants expressing the bar gene product PAT, were selected with GA.
Bayer characterized the genetic material introduced into transformation event LLCotton25 using genomic DNA blot analysis (also known as Southern blot analysis) and Polymerase Chain Reaction (PCR) amplification of genomic DNA. The results of these analyses are detailed in the August 30, 2002 submission and in the amendment dated January 8, 2003. Collectively, the data are consistent with a single insertion of a single copy of the transgene cassette, P35S-bar-3'nos into the genome of the LLCotton25 event. Additionally, there was no evidence of integration of the vector backbone into the cotton genome. Bayer also assessed the stability of the P35S-bar-3'nos transgene insertion over multiple generations and in different genetic backgrounds. Using genomic DNA blot analysis of NcoI digested DNA, the same restriction pattern was obtained upon hybridization of the blots with the complete T-DNA insertion, confirming the stable inheritance of the inserted DNA.
Bayer characterized the heritability of the transgene using standard Mendelian genetic tests. These genetic tests are detailed in the August 30, 2002 submission and the January 8, 2003 amendment. Bayer performed Chi-square analysis of the segregants to show a stable pattern of inheritance as expected for a single integrated transgene.
Bayer determined the levels of PAT protein present in both raw agricultural commodities and processed fractions of transformation event LLCotton25. Bayer used the enzyme linked immunosorbent assay (ELISA) technique. Polyclonal antisera raised against PAT protein was used in the ELISA to detect both inactive and enzymatically active PAT.
For the raw agricultural commodities, Bayer performed ELISAs on three fractions, cleaned seed, lint coat, and lint. Due to sample processing limitations, fuzzy seed was not analyzed directly, but separated into two fractions that were tested, cleaned seed and lint coat. The sum of PAT protein in these two fractions is equivalent to the amount of PAT protein in fuzzy seed. Bayer notes that more than 98.5% of the PAT protein is found in the cleaned seed fraction and therefore would be present in the fuzzy seed fraction. The lint coat and lint fractions contain less than 1.5% of the PAT protein. Bayer notes that PAT protein was approximately 0.029% and 0.003% of crude protein for fuzzy seed and lint, respectively. For ELISAs, Bayer used tissue from fields sprayed with Liberty® or a conventional herbicide regime.
For the processed commodities, Bayer performed ELISAs on extracts from fractionated agricultural products of cotton. All material originated from the field, under typical commercial growing conditions and was either sprayed with Liberty®, or untreated. PAT protein was found in all fractions of transgenic seed cotton and delinted cottonseed and in all transgenic samples of linters, cottonseed hulls, solvent extracted meal and toasted meal. PAT protein was not detected in transgenic samples of crude oil and deodorized oil or in any of the non-transgenic samples.
Bayer also provided data and information on the allergenic and toxic potential of the PAT protein. Bayer assessed the potential allergenicity of PAT protein from four different perspectives: in silico, in vitro, ex vivo and in vivo. Using the in silico approach, Bayer compared the amino acid sequence of PAT to known toxins and allergens in public databases and assessed the potential glycosylation sites. The result of these searches turned up no sequence similarities to known allergens or toxins and no potential glycosylation sites. To further address the allergenicity potential of PAT protein, Bayer cites published and unpublished proteolytic stability test studies. Bayer notes that ex vivo digestibility studies in pig and cattle stomach fluids indicate a rapid inactivation of PAT protein (less than one minute). In vitro digestibility tests in simulated gastric and intestinal fluids similarly show a rapid inactivation and degradation of the PAT protein. Bayer concludes from these experiments that the potential for PAT protein to survive the digestive tract and be absorbed is greatly minimized by the rapid degradation of the PAT protein, thus it is unlikely to elicit an allergic or toxic reaction. Finally, Bayer cites the results of both acute intravenous and subchronic oral, toxicity studies in rodents. These studies show no adverse or toxic effects.
To assess whether any unexpected modifications occurred in transformation event LLCotton25, Bayer compared the composition of raw commodities and processed fractions from both transgenic cotton, transformation event LLCotton25, and its non-transgenic counterpart, Coker 312.
In 2000, Bayer conducted six field trials under conditions typical of U.S. production practices in North Carolina, Arkansas, Mississippi, and Texas. There were six transgenic (transformation event LLCotton25) and three non-transgenic (Coker 312) plots at each site. Three of the six transgenic plots were treated twice with Liberty®, and three received a conventional herbicide regime. The cotton bolls were harvested and processed at cotton gins to obtain the long textile fiber lint and the cottonseed. Bayer analyzed proximates (crude protein, fat (ether extract), ash, crude fiber, neutral detergent fiber, acid detergent fiber, carbohydrates (by difference) and moisture) in ginned cottonseed and lint. Bayer also measured the following in ginned cottonseed only: amino acid and fatty acid composition, levels of antinutrients, key minerals, and Vitamin E (alpha tocopherol). Amino acids measured and reported included alanine, arginine, aspartic acid, cystine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Fatty acids measured and reported were: myristic, palmitic, stearic, arachidic, behenic, lignoceric, palmitoleic, oleic, linoleic, and linolenic. The three known antinutrients in cotton measured were: phytic acid, cyclopropenoid fatty acids (sterculic, malvalic, and dihydrosterculic), and gossypol (total and free). Key minerals measured were: calcium, phosphorus, potassium, iron, magnesium, and zinc. Bayer reports that the mean levels of antinutrients, key minerals, Vitamin E, and proximates in addition to the amino acid and fatty acid composition, revealed no significant differences between the transgenic and non-transgenic sample sets for cottonseed, or lint (where measured). In sum, no significant differences were detected in the composition of the transgenic raw commodities versus the composition of the non-transgenic raw commodities. Bayer's measured values are also within the literature range for these commodities as reported in this notification or available in other notifications.
Bayer further processed cotton from their North Carolina site into delinted cottonseed, linters, cottonseed meal, toasted cottonseed meal, hulls, crude oil and deodorized oil. All samples were obtained from field-grown cotton and included transgenic unsprayed, transgenic sprayed, and non-transgenic plants.
For the delinted cottonseed, cottonseed meal and toasted cottonseed meal, Bayer measured proximates, amino acids, key minerals, and antinutrients, all as detailed above. Linters were analyzed for proximates. Delinted cottonseed, crude oil, and deodorized oil were analyzed for fatty acids and either alpha tocopherol or total tocopherol. Delinted cottonseed, cottonseed meal, and toasted cottonseed meal were analyzed for total gossypol, free gossypol, phytic acid, and cyclopropenoid fatty acids while crude oil and deodorized oil were analyzed for total gossypol and cyclopropenoid fatty acids. Bayer summarizes the analyses performed for each processed commodity in Table VII.11 within their notice as well as providing the measured values for each analysis. The analyses Bayer performed were appropriate for a given commodity. For example, Bayer measured fatty acids, gossypol (total), and cyclopropenoid fatty acids in both crude and deodorized oil. The fatty acid profile reported for crude oil and deodorized oil are within the reported literature ranges. As only one sample was analyzed for each of the processed cotton products, the data do not lend themselves to statistical evaluation. However, there are not large differences in the values reported for the control cottonseed products versus the bioengineered cottonseed products with the exception of neutral detergent fiber (NDF) reported for toasted cottonseed meal. Bayer's January 8, 2003 amendment includes additional NDF data for toasted cottonseed meal that provides support that the original NDF value for control cottonseed meal was an aberrant result. Where there are literature values for the reported processed products, the values for the measured parameters fall within the literature range with the exception of some of the amino acids measured in the control cotton line. Low values are reported for the control cottonseed meal which may be due in part to the higher amount of fat and lower amount of crude protein remaining in the meal versus that reported in the literature. The amino acid values for the bioengineered cotton were at or above values reported in the literature.
Bayer has concluded that transformation event LLCotton25 is not materially different in composition, safety, or any other relevant parameter from cotton now grown, marketed, and consumed. At this time, based on Bayer's data and information, the agency considers Bayer's consultation on transformation event LLCotton25 to be complete.
/s/
Susan J. Carlson, Ph.D.
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