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U.S. Department of Health and Human Services

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Biotechnology Consultation Note to the File BNF No. 000092

Return to inventory: Completed Consultations on Foods from Genetically Engineered Plant Varieties

See also Biotechnology: Genetically Engineered Plants for Food and Feed and about Submissions on Bioengineered New Plant Varieties


Date: July 28, 2004

Subject: B.t. Cry1Ac insect resistant cotton event 3006-210-23

Keywords: Cotton, Gossypium hirsutum L., insect-protected cotton, Cry1Ac protein, phosphinothricin acetyltransferase (PAT), Agrobacterium tumefaciens, Bacillus thuringiensis subspecies kurstaki, Lepidopteran insects, cotton bollworm, pink bollworm, tobacco budworm.

Background

In a submission dated March 18, 2003, Mycogen Seeds/Dow AgroSciences LLC (DAS) provided to FDA a summary of the safety and nutritional assessment they conducted on the new bioengineered insect-protected cotton line, Cry1Ac cotton event 3006-210-23. The company provided additional information on May 30, June 27, September 5, and September 25, 2003. DAS concluded that the new insect resistant cotton is as safe and nutritious as conventional cotton varieties currently being marketed.

Intended Effect

The intended effect of this genetic modification of cotton is to confer resistance to Lepidopteran insect pests such as the cotton bollworm, pink bollworm, and tobacco budworm. To accomplish this objective, DAS introduced the cry1Ac gene from Bacillus thuringiensis (B.t.) subspecies kurstaki strain HD-73 and the pat gene isolated from Streptomyces viridochromogenes into the proprietary cotton Acala germplasm line known as GC510. The Cry1Ac cotton event 3006-210-23 is toxic to Lepidopteran insects. The pat gene confers tolerance to chemically synthesized phosphinothricin products such as glufosinate-ammonium and is used as a selectable marker. The Cry1Ac cotton event 3006-210-23 is intended to be marketed and sold as a stacked event product along with Cry1F cotton event 281-24-236, submitted in a separate filing (BNF 85) with FDA.

Regulatory Considerations

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 Cry1Ac protein in cotton event 3006-210-23 is considered a pesticidal substance and the phosphinothricin acetyltransferase (PAT) protein is considered an inert ingredient. Therefore, the safety assessment of these proteins falls under the regulatory purview of EPA.

Genetic Modifications and Characterization

DAS used the Acala cotton variety GC510 in the transformation of cotton event 3006-210-23. DAS used a disarmed Agrobacterium tumefaciens methodology for their transformation (Ooms et al., 1982; Zambryski, 1988). The T-DNA region of the plasmid vector pMYC3006 contains the elements listed in Table 1.

Table 1: Genetic elements contained in the T-DNA region of the plasmid vector pMYC3006
Genetic Element Description
Ubi Zm1 Zea mays promoter system for cry1Ac expression.
cry1Ac Synthetic, plant optimized, full length version of cry1Ac from B. thuringiensis subspecies kurstaki strain HD-73. Confers resistance to Lepidopteran insects.
ORF25 polyA Bi-directional terminator from A. tumefaciens strain LBA 4404 pTi15955 (Barker et al., 1983) for cry1Ac and pat gene.
pat Synthetic plant-optimized glufosinate-ammonium resistance gene, based on a phosphinothricin acetyltransferase gene sequence from Streptomyces viridochromogenes. Used as a selectable marker.
(4OCS)Δmas 2' Mannopine synthase promoter from A. tumefaciens strain LBA 4404 pTi15955 for pat expression.

The pMYC3006 transformation vector is a binary T-DNA vector carrying the transgenes for insertion into the plant genome between consensus T-DNA border sequences (from A. tumefaciens pTi15955 (Barker et al., 1983)), and a bacterial antibiotic resistance marker in the plasmid backbone, to facilitate cloning and maintenance of the plasmid in bacterial hosts. The plasmid backbone was derived from plasmid RK2 (Schmidhauser and Helsinki, 1985), from which the tetracycline resistance gene was deleted and replaced with a DNA fragment containing the erythromycin resistance gene for bacterial expression. DAS states that a lack of binding of the erythromycin probe to the expected DNA fragments indicates that the gene encoding for erythromycin resistance on plasmid pMYC3006 has not been integrated into the cotton genome for event 3006-210-23.

DAS characterized the integration and number of insertions in event 3006-210-23, using restriction enzyme digestion and Southern blot analysis. The results suggest a single, intact insertion of cry1Ac, pat, and the regulatory elements in event 3006-210-23.

DAS assessed the genetic stability of the inserted DNA within and across generations using Southern blot and segregation analysis. DAS states that the integration is stable and that the segregation of transgenes in event 3006-210-23 shows a Mendelian inheritance pattern.

Food/Feed Use

Cotton, Gossypium hirsutum L., is grown worldwide primarily as a source of fiber for textile manufacturing. Cottonseed, which is a by-product of the fiber production, is used in human food and animal feed production. Cottonseed contains the natural toxicants gossypol and cyclopropenoid fatty acids (CPFAs). Whole cottonseed is used as feed, supplying energy and fiber for dairy cows.

Alternatively, cottonseed can be processed into linters, hulls, oil and meal. As a first step in cotton processing the linters are removed. Linters are short fibers that are further 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. Next the hull, a protective seed coating, is removed. Hulls are used as a fiber component of livestock feeds due to their high cellulose and lignin content. Seeds without hulls are referred to as kernels or meat. The kernels are used for oil extraction. Cottonseed oil is routinely used in human food and has a long history of safe use. Cottonseed oil intended for human consumption is highly purified. The purification process substantially reduces the content of CPFAs. The refined cottonseed oil is used as frying oil, salad and cooking oil, and in various foods including mayonnaise, salad dressing, shortening, and margarine.

Cottonseed meal is the product obtained by finely grinding the flakes or cake which remain after removal of most of the oil from cottonseed by either a solvent or mechanical extraction process. Cottonseed meal is principally used as protein containing feed for livestock.

Compositional Analysis

DAS analyzed the composition of cotton event 3006-210-23 to assess whether any unexpected modifications occurred. DAS used null plants for their comparative compositional analysis. DAS describes these null plants as the F1 generation of plants lacking all transgenes after crossing the Cry1Ac and Cry1F BC3F1 lines to generate the stacked product. The null plants and the Cry1Ac event 3006-210-23 BC3F1 line were self-pollinated to the F4 generation to provide the seed source for the nutritional composition study. DAS states that the background of the null control is very closely related to the Cry1Ac event because it was derived from a late (BC3F1) generation of the transgenic event. DAS further states that the null control is also very closely related to the commercial variety, PSC355, because it has been backcrossed to the PSC355 variety four times, yielding a genetic background comprised of approximately 93.75% of the cotton variety PSC355. DAS concludes that the null plants represent the genetic background of both the Cry1Ac event and a commercial cotton variety.

The following groups of analytes were measured:

  • proximates
  • minerals
  • amino acids
  • fatty acids
  • anti-nutrients

A list of specific analytes contained in each group is shown in Table 2.

Analyses were performed on cottonseed, terminal leaf, square, and processed cottonseed including kernel, hull, toasted meal, and refined oil. DAS tested cotton grown at six different locations. DAS analyzed cottonseed samples individually and averaged the results across all six production sites. DAS compared mean values of the transgenic cotton to the non transgenic (null) control cotton line using a mixed model analysis of variance with p values being determined by a paired 't' test and Dunnett's procedure to maintain the experiment-wide error at 0.05. This procedure was utilized because in addition to the null control and event 3006-210-23, the experiment included event 281-24-236. Mean values were also compared to literature values. DAS analyzed processed cottonseed products by combining samples from all six sites into a single sample per treatment. Terminal leaf and square samples were reported individually from two locations.

Table 2. Compositional analytes
Proximates Minerals Amino Acids Fatty Acids Anti-Nutrients Anti-Oxidants
ash
total fat
moisture
crude protein
carbohydrates
crude fiber
acid detergent fiber (ADF)
neutral detergent fiber (NDF)
calories
calcium
copper
iron
magnesium
manganese
molybdenum
phosphorus
potassium
sodium
sulfur
zinc
 
aspartic acid
threonine
serine
glutamic acid
proline
glycine
alanine
cysteine
valine
methionine
isoleucine
leucine
tyrosine
phenylalanine
histidine
lysine
arginine
tryptophan
 
caprylic (8:0)
capric (10:0)
lauric (12:0)
myristic (14:0)
myristoleic (14:1)
pentadecanoic (15:0)
pentadecenoic (15:1)
palmitic (16:0)
palmitoleic (16:1)
heptadecanoic (17:0)
heptadecenoic (17:1)
stearic (18:0)
oleic (18:1)
linoleic (18:2)
gamma-linolenic (18:3)
linolenic (18:3)
arachidic (20:0)
eicosenoic (20:1)
eicosadienoic (20:2)
eicosatrienoic (20:3)
archidonic (20:4)
behenic (22:0)
 
gossypol-free
gossypol-total
cyclopropenoid
fatty acids
(malvalic, sterculic, dihydrosterculic acids)
polyphenols-total
aflatoxins B1, B2, G1, G2
 
alpha, beta, gamma, delta tocopherol

1. a     Raw commodity (cotton seed)

DAS determined the levels of the following components of cotton seed:

  • proximates
  • minerals
  • amino acids
  • fatty acids
  • anti-nutrients (CPFAs, gossypol-total)

DAS reports no significant differences for proximates between the control and the transgenic line except for crude fiber, where a significantly (p < .05) lower level was found in the transgenic sample compared to the control. The level for crude fiber was still within the published literature range. DAS also reports a significantly higher level for stearic fatty acid in the transgenic sample compared to the control, but the levels for both the control and transgenic cottonseed were below the range of literature values and hence are not considered an effect of the transgene. DAS reports no significant differences for minerals between control and transgenic samples, but reports sulfur levels slightly higher than literature values for both control and transgenic samples. These differences are not considered to be biologically meaningful. The remainder of the levels of proximates, minerals, and amino acids and anti-nutrients were not significantly different between the control and the transgenic line and were within literature ranges.

DAS determined the level of CPFAs. Cyclopropenoid fatty acids, which occur naturally in cottonseed, are considered to be anti-nutritional compounds, undesirable in human food and animal feed. They are known to inhibit the desaturation of stearic acid to oleic acid in the human or animal body and result in alteration of membrane permeability or an increase in the melting point of fats. In cottonseed oil intended for human consumption, the CPFAs are reduced to negligible levels.1 DAS reports no statistically significant differences between control line and transgenic line for CPFAs in cotton seed, and all values were within literature values.

DAS measured gossypol in cottonseed. Gossypol is a terpenoid aldehyde that naturally occurs in cottonseed and is toxic to humans and animals. DAS reports no statistically significant differences between control line and transgenic line, and all values were within literature values.

DAS measured four major aflatoxins, B1, B2, G1, and G2, in cottonseed. Aflatoxins are mycotoxins produced by certain species of the fungus Aspergillus that may infect cotton, mainly A. flavus and A. parasiticus. Aflatoxins are highly-substituted coumarins containing a fused dihydrofuran and are endemic to cotton grown in the southwestern U.S. Aflatoxins are potent animal toxins and carcinogens and have been epidemiologically implicated as environmental carcinogens in humans. DAS reports that these aflatoxins were not detected in either the control or transgenic cotton line.

1. b     Raw Commodity (terminal leaf and square)

DAS determined the levels of the following components of leaf and square tissue:

  • anti-nutrients (gossypol-total, polyphenols)

DAS measured polyphenols and gossypol in terminal leaf and square from two locations and found comparable results between the transgenic cotton and the controls, except for the lower gossypol levels in the control line compared to the transgenic line at one field site which DAS observed to be site specific.

2.        Processed commodity (cottonseed oil, kernels, toasted meal, hulls)

DAS analyzed kernels, hulls, toasted meal, and refined oil from single samples from bulk processing across all six production sites.

2. a     Cottonseed oil

DAS determined the levels of the following components of refined cottonseed oil:

  • proximates (fat, protein, moisture)
  • fatty acids
  • anti-nutrients (gossypol, free and total, CPFAs)
  • anti-oxidants

DAS states that analysis results for proximates and fatty acids were comparable between the transgenic line and the control line and that values fell within reported literature ranges. DAS reports similar results for control and transgenic lines for both gossypol and CPFAs. No free gossypol was detected and low total gossypol levels were found in the samples analyzed. The levels of CPFAs were also similar for control and transgenic samples and were within literature values.

Cottonseed oil was also analyzed for tocopherol isomers. Tocopherols, which may function as naturally occurring antioxidants, showed similar results for control and transgenic lines and fell within Codex Alimentarius standards for occurrence of alpha, beta, gamma, and delta tocopherols in crude cotton seed oil. Oil processing steps subsequently remove tocopherols from the oil.

2. b    Kernels

DAS determined the levels of the following components in kernels:

  • proximates (moisture)
  • anti-nutrients (gossypol, free and total)

Proximate analysis was comparable for the Cry1Ac event 3006-210-23 line and control line, and all values were within reported literature values. Anti-nutrient analysis of the kernel was also similar for the control line and the transgenic line. Literature values for anti-nutrients in kernels were not available.

2. c    Toasted meal

DAS determined the levels of the following components of toasted meal:

  • proximates
  • minerals
  • amino acids
  • anti-nutrients (gossypol, free and total)

Analysis results were comparable for all parameters tested between the control and the transgenic line and were near or within literature values.

2. d    Hulls

DAS determined the levels of the following components in hulls:

  • proximates (ash, fat, moisture, protein, and carbohydrates)
  • minerals

Analysis results were comparable for the Cry1Ac event 3006-210-23 and the control line, and values fell within literature ranges.

Conclusions

DAS has concluded that the Cry1Ac cotton event 3006-210-23 is not materially different in composition, safety, wholesomeness, or any relevant parameter from cotton now grown, marketed, and consumed. At this time, based on DAS's data and information, the agency considers DAS's consultation on Cry1Ac cotton event 3006-210-23 to be complete.

 

Karin Ricker, Ph.D.


 

References

Barker, R., K. Idler, D. Thompson and J. Kemp. 1983. Nucleotide sequence of the T-DNA region from the Agrobacterium tumefaciens octopine Ti plasmid pTil5955. Plant Mol. Biol. 2:335-350.

Ooms, G., P. Hooykaas, R. V. Veen, P. V. Beelen, T. Regensburg-Tuink and R. Schilperoort. 1982. Octopine Ti-plasmid deletion mutants of Agrobacterium tumefaciens with emphasis on the right side of the T-region. Plasmid. 7: 15-29.

Schmidhauser, T.J. and Helinski, D.R. 1985. Regions of broad-host range plasmid RK2 involved in replication and stable maintenance in nine species of gram-negative bacteria. J. Bacteriol. 164(1), 446-455.

Zambryski, P. 1988. Basic processes underlying Agrobacterium-mediated DNA transfer to plant cells. Ann. Rev. Genet. 22: 1-30.


(1)"Cottonseed Oil." In "Bailey's Industrial Oil & Fat products." 1996. John Wiley & Sons, Inc. Vol. 2, Chapter 4, pp. 159-240.