Biotechnology Consultation Note to the File BNF No. 000112
Return to inventory: Submissions on Bioengineered New Plant Varieties
Date: February 6, 2009
Subject: Biotechnology Notification File (BNF) 000112, cotton transformation event COT67B.
Keywords: Gossypium hirsutum L., cotton, Event COT67B, SYN-IR67B-1, Bacillus thuringiensis, FLCry1Ab, flcry1Ab, full-length Cry1Ab, Lepidopteran insect resistance, Agrobacterium tumefaciens
In a submission dated July 3, 2007, Syngenta Seeds, Inc. (Syngenta) provided to the Food and Drug Administration (FDA) a safety and nutritional assessment of genetically engineered cotton designated as cotton event COT67B. Syngenta provided additional information to the FDA on December 13, 2007 and December 8, 2008. Syngenta concluded that food and feed derived from cotton event COT67B are as safe and nutritious as food and feed derived from conventional cotton varieties currently being marketed.
2 Intended Effect
The intended effect of the modification in cotton event COT67B is to confer resistance to a number of Lepidopteran insects. To accomplish this objective, Syngenta introduced the full-length cry1Ab (flcry1Ab) gene (derived from Bacillus thuringiensis subsp. kurstaki strain HD-1) into the conventional cotton cultivar Coker 312 and designated the resulting cotton event as COT67B. The FLCry1Ab protein, encoded by the flcry1Ab gene, confers resistance to Lepidopteran insects, including cotton bollworm, tobacco budworm, pink bollworm, and cabbage looper.
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 flcry1Ab gene in cotton event COT67B and resulting expression products are considered pesticidal substances. EPA considers the recombinant DNA constructs used in the development of cotton event COT67B to be part of the PIP in cotton event COT67B, and therefore EPA is reviewing the recombinant DNA constructs and resulting expression products.
4 Genetic Modifications and Characterizations
To generate cotton event COT67B, Syngenta used an Agrobacterium tumefaciens-mediated transformation system. Syngenta transformed petioles of conventional cotton cultivar Coker 312 using two plasmid vectors, pNOV4641 and pNOV1914. The T-DNA region of pNOV4641 contains a single copy of the flcry1Ab gene derived from B. thuringiensis subsp. kurstaki strain HD-1. The T-DNA region of pNOV1914 contains a single copy of the selectable marker gene aph4, which encodes hygromycin-B phosphotransferase. Expression of hygromycin-B phosphotransferase confers resistance to the antibiotic hygromycin, which was used for selection of transformed tissue during the initial tissue culture phase of transformation. However, once transformants were identified, Syngenta used traditional breeding techniques to select plants containing flcry1Ab but not the aph4 gene from pNOV1914 or elements from its plasmid backbone. Syngenta reports that the aph4 gene and related regulatory sequences are no longer present in cotton event COT67B.
|RB (right border)||Right border region of T-DNA from A. tumefaciens nopaline Ti-plasmid.|
|Act2||Promoter region from the actin-2 gene of Arabidopsis thaliana and its intron.|
|flcry1Ab||The full-length cry1Ab gene encoding the FLCry1Ab protein.|
|NOS||Terminator sequence from the nopaline synthase gene of A. tumefaciens.|
|LB (left border)||Left border region of T-DNA from A. tumefaciens nopaline Ti-plasmid.|
Syngenta used restriction enzyme digestion of genomic DNA followed by Southern blot analysis to characterize the inserted DNA in cotton event COT67B, and confirmed this characterization by DNA sequence analysis of the entire insert. Syngenta states that based on the results obtained from these analyses, the T-DNA from plasmid pNOV4641 is present as a single intact copy in cotton event COT67B with the exception of some truncation at the right and left T-DNA borders that does not affect functionality. Southern blot analysis of cotton event COT67B did not reveal the presence of the aph4 gene and associated regulatory elements derived from pNOV1914, nor any of the backbone sequences from either transformation plasmid (pNOV4641 and pNOV1914). Sequence analysis of the cotton genome sequences flanking the insert did not indicate disruption of any known endogenous cotton gene, and revealed no novel open reading frames spanning either the 5' or 3' junctions between the insert and the cotton genome. Syngenta also assessed genetic stability of the flcry1Ab gene in cotton event COT67B over several generations and concluded that the transgenic locus is stable and inherited in Mendelian fashion as a single locus dominant trait.
5 Food and Feed Uses of Cotton
Whole ginned cottonseed (‘fuzzy seed’) and meal are consumed as animal feed, and other processed fractions are consumed as human food. The whole seed is a source of protein, fat, and energy in feed for dairy and beef cattle as well as sheep. Refined cottonseed oil is the major cotton by-product consumed by humans, although small quantities of cottonseed linters are processed into cellulose and used in some human foods such as sausage casings and salad dressing.1
6 Compositional Analysis
Syngenta tested the composition of whole cottonseed from cotton event COT67B and the non-transgenic variety Coker 312 (the control) and also examined certain processed cottonseed fractions.
6.1 Whole Cottonseed
Syngenta conducted a study to obtain compositional data on whole cottonseed samples of cotton event COT67B and Coker 312 control cotton. Four field trials were conducted under current production practices in typical cotton-producing areas of the southern United States. There were four cotton event COT67B and four Coker 312 control cotton plots at each test site. A sample of whole cottonseed was taken from each of the field plots.
Syngenta reports the levels of 43 different components from samples of whole cottonseed. Syngenta analyzed cottonseed for proximates, amino acids, fatty acids, minerals, and other analytes of interest. A list of specific analytes is shown in Table 2.
An analysis of variance (ANOVA) was performed for all but two analytes. A significance level of p ≤ 0.05 (a ≤ 5%) was chosen.
|Proximates||Minerals||Amino Acids||Fatty Acids||Anti-nutrients and|
acid detergent fiber
neutral detergent fiber
total dietary fiber
|Vitamin E (α-tocopherol)|
|aThese fatty acids were near the limit of quantitation (0.02 – 0.06% fw)|
Syngenta made the following conclusions regarding the results of the levels of analytes detected in whole cottonseed.
- Proximates: Syngenta found no statistically significant differences in proximate composition between cotton event COT67B and Coker 312 control cotton. The mean levels for each of these analytes were within the range of values reported in either the International Life Science Institute (ILSI) Crop Composition Database (ILSI 2006), the Organisation for Economic and Co-operation and Development’s “Consensus Document on Compositional Considerations for New Varieties of Cotton (Gossypium hirsutum and Gossypium barbardensis): Key Food and Feed Nutrients and Anti-Nutrients” (OECD, 2004), or both (hereafter referred to as publicly available references).
- Minerals: Syngenta found no statistically significant differences in phosphorous content between cotton event COT67B and Coker 312 control cotton. There was a statistically significant increase in calcium content in COT67B. However, the magnitude of the difference was small and mean levels of calcium for both treatments were within the range of values reported in publicly available references.
- Amino Acids: Syngenta found no statistically significant differences between cotton event COT67B and Coker 312 control cotton for any of the 18 amino acids measured. The mean levels of these amino acids were within the range of values reported in publicly available references.
- Fatty Acids: Syngenta found statistically significant differences in the mean levels of three fatty acids between cotton event COT67B and Coker 312 control cotton samples. Palmitic acid was higher as a percentage of total fatty acids in COT67B than in Coker 312, whereas stearic and oleic acids were lower. However, the magnitude of the differences was small, and the mean levels for all seven major fatty acids were within the range of values reported in publicly available references. Syngenta reported levels of behenic acid (22:0; 0.02-0.04% fresh weight (fw)) and arachidic acid (20:0; 0.03-0.06% fw) that were very low and close to the limit of quantitation; these analytes were excluded from the statistical analyses. Syngenta also reported that a number of other fatty acids were below the limit of quantification.2
- Anti-Nutrients: Syngenta found that the relative amount of dihydrosterculic acid as a percentage of total fatty acids was lower in cotton event COT67B than Coker 312 control cotton. Levels of all the cyclopropenoid fatty acids, including dihydrosterculic acid as well as total and free gossypol levels, were within the range of values reported in publicly available references.
- Vitamin E (α-tocopherol): Syngenta found that Vitamin E levels were statistically significantly higher in cotton event COT67B than in Coker 312 control cotton. However the magnitude of the difference was small and the mean values for both COT67B and Coker 312 were within the range of values reported in publicly available references.
6.2 Processed Cottonseed Fractions
Samples of cottonseed from Coker 312 control cotton and cotton event COT67B from one of the field locations were processed into toasted meal and refined cottonseed oil. Analyses were performed on two samples of oil (total gossypol as well as the cycloproprenoid fatty acids: sterculic acid, malvalic acid and dihydrosterculic acid) and one sample of meal (total and free gossypol). Syngenta concluded that there was no evidence of a biologically significant difference in gossypol or the measured cyclopropenoid fatty acids in toasted meal or refined oil produced from cotton event COT67B relative to Coker 312 control cotton.
Syngenta has concluded that Lepidopteran-insect resistant cotton event COT67B, and foods and feeds derived from it, are not materially different in safety, composition, or any other relevant parameter from cotton now grown, marketed, and consumed. At this time, based on Syngenta’s data and information, the agency considers Syngenta’s consultation on cotton event COT67B to be complete.
1FDA has also approved a number of modified cottonseed products as food additives for human consumption (21 CFR 172.894).
2Other fatty acids were reported to be below the limit of quantitation (0.01-0.02% fw): caprylic (8:0), capric (10:0), lauric (12:0), myristoleic (14:1), pentadecanoic (15:0), pentadecenoic (15:1), heptadecanoic (17:0), heptadecenoic (17:1), γ-linolenic (18:3), eicosenoic (20:1), eiconsadienoic (20:2), eicosatrienoic (20:3), and arachidonic (20:4) acids.