Biotechnology Consultation
Note to the File
BNF No.
000074
Date: July 16, 2002
Subject: Insect-Protected Bollgard II Cotton Line 15985
Keywords: Cotton, Gossypium hirsutum L., insect-protected cotton, Bollgard II cotton, Cry2Ab protein, Cry1Ac protein, GUS protein, neomycin phosphotransferase type II protein, Bacillus thuringiensis subsp. kurstaki, lepidopteran insects, cotton bollworm, tobacco budworm, pink bollworm, armyworm.
In a submission dated June 29, 2000, Monsanto Company submitted to FDA a summary of the safety and nutritional assessment they have conducted on the new bioengineered insect-protected Bollgard II cotton line 15985. The company provided additional information in submissions dated December 18, 2000; August 10, 2001; February 4, 2002; and March 12, 2002. Monsanto concluded that the cotton line 15985 is as safe and nutritious as conventional cotton varieties currently being marketed. Monsanto had previously completed consultations with FDA on insect-protected cotton lines 531, 757, and 1076 (BNF 000013).
The intended effect of this genetic modification of cotton is to confer resistance to lepidopteran insect pests such as the cotton bollworm, tobacco budworm, pink bollworm, and armyworm. To accomplish this objective, Monsanto introduced the cry2Ab gene from Bacillus thuringiensis subsp. kurstaki into the cotton line DP50B already containing the cry1Ac gene from B. thuringiensis subsp. kurstaki. The cry1Ac and cry2Ab genes encode the Cry1Ac and Cry2Ab proteins, respectively. Both proteins are toxic to lepidopteran insects. Monsanto also introduced into the DP50B genome the uidA gene that encodes the GUS protein. The GUS protein serves as a scorable marker in the identification of the plant cells transformed with the cry2Ab gene.
The Environmental Protection Agency (EPA) regulates plant-incorporated protectants under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) in 40 CFR Parts 152 and 174. According to EPA regulations, "Plant-incorporated protectant means a pesticidal substance that is intended to be produced and used in a living plant, or in the produce thereof, and the genetic material necessary for production of such a pesticidal substance. It also includes any inert ingredient contained in the plant, or produce thereof" (40 CFR 152.3). Therefore, the Cry2Ab protein and the genetic material necessary for its production are pesticidal substances and the GUS protein is an inert ingredient. As such, these substances are under the EPA jurisdiction. Although FDA has summarized the information on the genetic material and the Cry2Ab and GUS proteins provided in Monsanto's biotechnology notification file (BNF 000074), the agency has not evaluated the information related to these substances.
According to the original submission (June 29, 2000), Monsanto submitted to EPA requests for exemption from the requirement of tolerances for Cry2Ab and GUS proteins in the fall of 1999. They also submitted to EPA a request for registration of the Cry2Ab protein as a plant-incorporated protectant in April, 2000. Monsanto also expressed their intent to submit to the United States Department of Agriculture (USDA) a request for the non-regulated status for the Bollgard II cotton line 15985 and the progenies derived by crosses of this cotton line with other cotton lines.
Cotton, Gossypium hirsutum L., is grown worldwide primarily as a source of fiber in the textile manufacturing. 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. The refined cottonseed oil is used as frying oil, salad and cooking oil, 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. The hull is a protective coating of the seed that is removed prior to processing cottonseed into oil and meal. Hulls are used as a fiber component of livestock feeds due to their high cellulose and lignin content.
Monsanto used Delta and Pine Land Company variety DP50B as the parental variety. A traditional cross between cotton variety DP50 and the bioengineered Bollgard cotton line 531 produced the DP50B variety. Bollgard cotton event 531 contains the cry1Ac and nptII genes that are expressed and the non-expressed aad bacterial marker gene. Therefore, the parental line of Bollgard 15985 (i.e., BP50B) also contains these genes and expresses the insecticidal Cry1Ac protein and the NPTII selectable marker protein.(1) Bollgard cotton line 531 was the subject of BNF 000013. Monsanto commercialized the cotton event 531 in 1996. The cotton varieties derived from the 531 event were grown in the United States on more than 3.9 million acres in 1999.
Monsanto constructed the plasmid vector PV-GHBK11 based on the widely used series of pUC plasmids. The vector backbone contains the pUC19 replication origin, which allows the replication of the vector in the intermediate host E. coli. The vector backbone also contains the nptII gene. The DNA segment of the PV-GHBK11 vector intended for transformation, designated PV-GHBK11L, contains the cry2Ab and uidA gene cassettes. The cry2Ab gene cassette consists of the enhanced 35S promoter from the cauliflower mosaic virus (CaMV), the synthetic cry2Ab coding region based on the sequence of the cry2Ab gene from B. thuringiensis (B.t.) subsp. kurstaki, and the 3' nontranslated region of the nopaline synthase gene from Agrobacterium tumefaciens that provides the transcription termination signal. The uidA gene cassette contains the E. coli uidA gene flanked by the same regulatory sequences as the cry2Ab gene in the cry2Ab gene cassette.
The PV-GHBK11 vector was propagated in E. coli and digested with the restriction enzyme KpnI to separate the backbone from the DNA segment PV-GHBK11L. The PV-GHBK11L segment was subsequently purified, precipitated onto gold particles, and introduced into the meristems of the recipient variety DP50B by particle acceleration. The transformed tissue was identified by histochemical staining that allows visual detection of the uidA gene product, the GUS protein.(2) The nontransformed tissue was gradually removed, thereby promoting the growth of meristems containing the introduced DNA. The seeds resulting from the GUS-positive plants were screened for the production of the Cry2Ab protein. Based on this screening, the transformant referred to as the cotton event 15985, was selected for commercial development.
Monsanto characterized the DNA inserted into Bollgard II cotton line 15985 using Southern blot analysis and polymerase chain reaction (PCR). The notifier assessed the stability of the inserted DNA across several generations using segregation studies and Southern blot analysis.
Monsanto tested the transgenic line 15985 for expression of the Cry2Ab and GUS proteins. Monsanto determined that the mean levels of the Cry2Ab and GUS proteins in cottonseed collected from eight field locations were 43.2±5.7 microgram/g and 58.8 microgram/g (fresh weight), respectively. Monsanto noted that cotton consumable fractions, oil and linters are both chemically and thermally processed and any proteins, including the Cry2Ab and GUS would be removed or denatured.
Monsanto stated that the Cry2Ab protein has a high degree of amino acid sequence similarity (97 percent) to the Cry2A protein, that is a component of various B.t. microbial products, and has been evaluated in numerous animal and human studies. Monsanto has also provided data and information on the allergenic and toxic potential of the Cry2Ab protein. Monsanto compared the amino acid sequence of the Cry2Ab protein with the sequences of known allergens and toxins and noted that the Cry2Ab does not share significant sequence similarity with these proteins. Monsanto examined the stability of the Cry2Ab protein in simulated gastric and intestinal fluids and its potential toxicity in an acute oral gavage study in mice. Monsanto performed a similar assessment of the GUS protein.
As noted in section 3 above, the Cry2Ab and GUS proteins are subject of EPA regulations. Therefore, although we have briefly summarized the information on these proteins provided by Monsanto, we have not evaluated this information.
To assess whether any unexpected modifications occurred in the cotton line 15985, Monsanto measured the levels of 48 components in cottonseed samples collected from eight field trials performed in six states in 1998. Monsanto measured each component in the transgenic line 15985, transgenic parent line DP50B, non-transgenic ancestral line DP50, four commercially available non-transgenic cotton varieties, and ten commercially available transgenic and non-transgenic cotton varieties. Monsanto reported the results as mean values and as ranges. Subsequently, Monsanto compared the level of each component in the transgenic line 15985 with the levels in the parent lines and the ranges determined for the commercial reference lines. Monsanto subjected the composition data obtained for the transgenic 15985 line and the transgenic parent DP50B line to statistical analysis.
Monsanto analyzed the seeds for proximates (protein, fat, carbohydrate(3), moisture, crude fiber, and calories), amino acids, fatty acids, and minerals (calcium, copper, iron, magnesium, manganese, phosphorus, potassium, sodium, and zinc). Monsanto reports that the mean levels of proximates, amino acids, and minerals in the 15985 line and the parent lines are not significantly different and are within the published ranges and the ranges determined by Monsanto for commercial reference varieties.
Monsanto measured the following fatty acids in cottonseed: myristic, palmitic, palmitoleic, stearic, oleic, linoleic, linolenic + gamma linoleic, arachidic, and lignoceric. Monsanto reports the level of each fatty acid as a percent of total fatty acids. According to Monsanto, there are no significant differences in the mean levels of palmitic, palmitoleic, oleic, linolenic + gamma linoleic, arachidic, and lignoceric acids between the 15985 and DP50B lines. Monsanto noted small but statistically significant differences in the mean levels of myristic, stearic, and linoleic acids. For example, the mean levels of linoleic acid in the 15985 and DP50B lines were 52.52 percent and 53.10 percent, respectively. The mean levels of all fatty acids in the 15985 and DP50B lines fell within the commercial cotton reference ranges. Therefore, Monsanto has not considered small differences in the levels of three fatty acids to be biologically relevant.
Monsanto also measured the levels of cyclopropenoid fatty acids (CPFAs), gossypol, and aflatoxins. As noted earlier (section 5.1), CPFAs and gossypol occur naturally in cottonseed. Aflatoxins are mycotoxins produced by certain species of the fungus Aspergillus that may infect cotton, mainly Aspergillus flavus and Aspergillus parasiticus. According to the literature cited by Monsanto, cottonseed is one of the commodities most commonly contaminated by aflatoxins.
Cyclopropenoid fatty acids are considered to be antinutritional 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.(4) Monsanto identified three CPFAs in the cottonseed, malvalic, sterculic, and dihydrosterculic. These acids were present in the 15985 line at mean levels of 0.45 percent, 0.30 percent, and 0.18 percent, respectively. Although these levels were higher than the corresponding levels in the DP50B variety (0.39 percent, 0.25 percent, and 0.15 percent)(5) they were well within the ranges determined by Monsanto for the reference commercial cotton varieties.
Gossypol is toxic to humans and animals. It is a terpenoid aldehyde that naturally occurs in cottonseed. Monsanto reports that there is no difference in the mean levels of gossypol in the 15985 line and the parent DP50B line and that these levels are within the range determined for the reference commercial cotton varieties.
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. Monsanto measured four major aflatoxins, B1, B2, G1, and G2, in cottonseed. The company reports that these aflatoxins were not detected in the 15985 line, DP50B line, and commercial reference lines at the detection limit of 1 ppb.
At the request of the Center of Veterinary Medicine, Monsanto has recently provided the levels of ADF (acid detergent fiber) and NDF (neutral detergent fiber) in cottonseed from the transgenic line 15985 and two control lines, DP50B and DP50 (submission of March 12, 2002). All three varieties were harvested from large scale field production plots in Argentina during the 1999/2000 growing season. The mean(6) NDF values were 52.8 percent and 52.7 percent and 52.6 percent for the 15985, DP50B, and DP50 varieties, respectively. Although these values were not significantly different from each other, they were slightly above the range of NDF values published in the literature for commercial cotton varieties.(7) The ADF mean values were 39.4 percent for line 15985, 37.5 percent for line DP50B, and 37.3 percent for line DP50. These values were not significanly different from each other and were within the range of ADF values published in the literature.
Monsanto pooled cottonseed samples collected by line across eight field sites, thereby creating one composite sample for each line. Each composite sample was processed into oil and analyzed for the following parameters: fatty acid composition, vitamin E content, gossypol content, and cyclopropenoid fatty acid content.
The fatty acid profiles of oil generated from the 15985 and DP50B lines were similar and consistent with the ranges determined for oil from commercial varieties. The level of vitamin E in the oil from the transgenic line 15985 was slightly above the level in the oil from the parent DP50B variety and the upper value obtained for the oil from commercial reference varieties. However, it was within the range reported in the literature (10.2 - 66.0 mg/100g).
Gossypol was not detected in any of the oil samples at the detection limit of 0.005 percent. The levels of cyclopropenoid fatty acids (sterculic, dihydrosterculic, and malvalic) in oil from the 15985 line were similar to those in the oil from the parental line and were within the range determined for oil derived from commercial varieties.
Toasted cottonseed meal samples were generated by pooling cottonseed across eight field sites for processing into a single sample per line. When cottonseed is processed into oil and meal, the lysigenous glands are ruptured and gossypol is released. Some of the gossypol binds to seed components. The bound gossypol is essentially inactive. In contrast, the free gossypol is considered toxic. Monsanto measured free gossypol and total (free and bound) gossypol in all samples of the toasted meal. The level of free gossypol in the meal from line 15985 was 0.037 percent (fresh weight) which was similar to the level in the meal from the parent DP50B line (0.042 percent). Free gossypol levels in meal samples from commercial varieties ranged from 0.025 to 0.068 percent.
The total gossypol level in cottonseed meal from lines 15985 and DP50B was 0.968 percent and 1.05 percent, respectively. Both levels are within the range determined for meal samples generated from the reference commercial cottonseed varieties.
Monsanto has concluded that Bollgard II transgenic cotton line 15985 is not materially different in composition, safety, or any other relevant parameter from cotton now grown, marketed, and consumed. At this time, based on Monsanto's data and information, the agency considers Monsanto's consultation on Bollgard II cotton line 15985 to be complete.
| /s/ Zofia S. Olempska-Beer, Ph. D. |
(1)The NPTII protein is also known as the APH(3')II protein. The protein is an enzyme, aminoglycoside 3'-phosphotransferase II. It catalyzes the phosphorylation and, thereby, inactivation of kanamycin, neomycin, gentamicin and other aminoglycoside antibiotics. The APH(3')II protein is an approved food additive for use in the development of bioengineered cotton, oilseed rape, and tomatoes in 21 CFR 173.170.
(2)The GUS protein is an enzyme beta-D-glucuronidase that catalyzes the hydrolysis of beta-glucuronides including the artificial substrate p-nitrophenyl-beta-D-glucuronide. Hydrolysis of this compound releases a blue dye that serves as a visible scorable marker in the plant transformation process.
(3)Carbohydrate values were calculated.
(4)"Cottonseed Oil." In "Bailey's Industrial Oil & Fat products." 1996. John Wiley & Sons, Inc. Vol. 2, Chapter 4, pp. 159-240.
(5)The mean levels of these acids in the 12985 line were statistically significantly different from the levels in the DP50B line (p<0.05).
(6)Based on 8 samples for lines 15985 and DP50 and 7 samples for line DP50B. All values are reported on a dry weight basis.
(7)FDA notes that the National Research Council (Nutrient Requirements of Dairy Cattle, 2001 and Nutrient Requirements of Beef Cattle, 1996; National Academy Press, Washington DC) reports NDF values from 50.3 percent to 51.6 percent, which are comparable to the values reported by Monsanto.