GM Crop Database

Database Product Description

DAS-06275-8 (DAS-Ø6275-8)
Host Organism
Zea mays (Maize)
Trait

Insect resistant, Lepidoptera.

Trait Introduction
Agrobacterium tumefaciens-mediated plant transformation.
Proposed Use

Production for human consumption and livestock feed.

Product Developer
DOW AgroSciences LLC

Summary of Regulatory Approvals

Country Food Feed Environment Notes
Canada 2006 2006 2006
Japan 2007 2008 2008
United States 2004 2004 2004

Introduction Expand

The maize line DAS-06275-8 (hereafter referred to as TC-6275) was genetically modified to contain two novel genes, cry1F and bar, for insect resistance and herbicide tolerance respectively. Both genes were introduced into the parental maize hybrid line Hi-II by Agrobacterium-mediated plant transformation.

The cry1F gene, isolated from the common soil bacterium Bacillus thuringiensis (Bt) var. aizawai, produces the insect control protein Cry1F, a delta-endotoxin. Cry proteins, of which Cry1F is only one, act by selectively binding to specific sites localized on the lining of the midgut of susceptible insect species. Following binding, pores are formed that disrupt midgut ion flow, causing gut paralysis and eventual death due to bacterial sepsis. Cry1F is lethal only when eaten by the larvae of lepidopteran insects (moths and butterflies), and its specificity of action is directly attributable to the presence of specific binding sites in the target insects. There are no binding sites for the delta-endotoxins of B. thuringiensis on the surface of mammalian intestinal cells, therefore, livestock animals and humans are not susceptible to these proteins. The Cry1F protein expressed in TC-6275 provides protection against European corn borer (ECB), southwestern corn borer (SWCB), fall armyworm (FAW), black cutworm (BCW), and some control of corn earworm (CEW).

The bar gene in TC-6275 confers tolerance to glufosinate ammonium. This herbicide can therefore be applied to TC-6275 as a weed control option, and can also be used as a selection tool for successful transformants containing the insect-tolerant cry1F gene. The herbicide tolerance of TC-6275 is due to the expression of a protein, phosphinothricin-acetyl-transferase (PAT), which confers tolerance to the active ingredient L-phosphinothricin in glufosinate ammonium. The bar gene was isolated from Streptomyces hygroscopius, a gram-positive soil bacterium.

Glufosinate ammonium is a post-emergence, broad-spectrum contact herbicide and plant dessicant. L-Phosphinothricin was first isolated as bialaphos, an antibiotic synthesized during the fermentation of Streptomyces hygroscopicus or S.viridochromogenes. In herbicidal formulations, L-Phosphinothricin is a component of the glufosinate ammonium, which is chemically-synthesized. Glufosinate ammonium is a racemic mixture of L-phosphinothricin, the herbicidal active moiety, and its D-enantiomer. L-phosphinothricin is structurally similar to glutamate, the substrate of glutamine synthetase (GS), an enzyme that catalyzes the synthesis of glutamine from glutamate and ammonia. L-phosphinothricin inhibits the activity of GS irreversibly by binding to its active sites. The inhibition of GS caused by the application of glufosinate ammonium to plants results in the accumulation of ammonia, the reduction in the levels of glutamine, and the inhibition of photosynthesis, all of which results in the death of the plant. Plants transformed with the bar gene express the enzyme phosphinothricin-acetyl-transferase (PAT) which acetylates L-phosphinothricin into a non-phytotoxic metabolite (N-acetyl-L-glufosinate).

Summary of Introduced Genetic Elements Expand

Code Name Type Promoter, other Terminator Copies Form
bar phosphinothricin N-acetyltransferase HT CaMV 35S ADH1 intron 1 potato pinII termination and poly(A) signal
mocry1F cry1F delta-endotoxin IR

ubiquitin 1 (Zea mays)

potato pinII termination and poly(A) signal

Characteristics of Zea mays (Maize) Expand

Center of Origin Reproduction Toxins Allergenicity

Mesoamerican region, now Mexico and Central America

Cross-pollination via wind-borne pollen is limited, pollen viability is about 30 minutes. Hybridization reported with teosinte species and rarely with members of the genus Tripsacum.

No endogenous toxins or significant levels of antinutritional factors.

Although some reported cases of maize allergy, protein(s) responsible have not been identified.

Donor Organism Characteristics Expand

Latin Name Gene Pathogenicity
Streptomyces hygroscopicus bar S. hygroscopicus is ubiquitous in the soil and there have been no reports of adverse affects on humans, animals, or plants.
Bacillus thuringiensis var. aizawai cry1F While target insects are susceptible to oral doses of Bt proteins, no evidence of toxic effects in laboratory mammals or birds.

Modification Method Expand

TC-6275 maize was produced by Agrobacterium-mediated transformation of the inbred maize line Hi-II with the plasmid vector PHP12537. The T-DNA segment of PHP12537 contained sequences of a modified (i.e., synthetic, maize-optimized and truncated) form of the cry1F gene (mocry1F) from Bacillus thuringiensis var. aizawai strain PS811, and the phosphinothricin N-acetyltransferase (PAT) encoding bar gene from Streptomyces hygroscopicus. The nucleotide sequence of the cry1F gene was modified to contain codons to optimize expression of the Cry1F protein in maize. Transcription of the cry1F gene was directed by the promoter, intron and 5' untranslated sequences from the maize ubiquitin gene. Terminator sequences were derived from the Solanum tuberosum (potato) proteinase inhibitor II (PINII). The expression of the bar gene was regulated by the 35S promoter and upstream enhancer sequences from the Cauliflower Mosaic Virus (CaMV) strain 1841, and the alcohol dehydrogenase intron (ADH1) from Zea mays. Terminator sequences were derived from the Solanum tuberosum proteinase inhibitor II.

Characteristics of the Modification Expand

The Introduced DNA

Southern blot analysis of the genomic DNA of TC-6275 demonstrated the integration of a single copy of the T-DNA of PHP12537. The analysis confirmed the intact insertion of the bar gene and its promoter and terminator sequences, as well as the cry1F gene and its terminator sequences; however, the analysis revealed that the promoter region of the cry1F gene transcription unit was truncated at the 5’ end. Cry1F was constitutively expressed despite the incomplete insertion of the promoter (v. Expressed Material). The sequences outside of the T-DNA border, including the spectinomycin resistance (spc) and tetracycline (tet) resistance genes, were not integrated into the genome of TC-6275.

Genetic Stability of the Trait

Southern blot analysis was also used to verify the stability of the T-DNA insert. The analysis demonstrated the stability of the insert containing the cry1F and bar genes, across three generations of TC-6275. Segregation analysis also demonstrated a stable insertion consistent with a Mendelian inheritance pattern for a dominant trait.

Expressed Material

The levels of expression of the Cry1F protein were quantified in various tissues of TC-6275 maize using enzyme-linked immunosorbent assay (ELISA) technique. Samples were taken from 6 field trials conducted at 3 locations in Chile from 2001 to 2002. Cry1F was found to be expressed throughout the plant and was quantified in samples collected at five growth stages (V9: vegetative, 9 leaves developed; R1: silking; R4: dough stage; physiological maturity; senescence). Mean levels of Cry1F, expressed on a dry weight basis, were: 16.7 ng/mg in leaves at V9; 6.22 and 7.17 ng/mg in the whole plant at V9 and R1, respectively; 11.0 ng/mg in stalks at R1; 6.26 ng/mg in forage at R4; and 2.47 ng/mg in the whole plant at senescence. Mean levels of Cry1F in pollen were 3.67 ng/mg, and 1.14 ng/mg in grain at physiological maturity.

Western blots of SDS-PAGE (sodium dodecylsulfate polyacrylamide) gel electrophoresis separated Cry1F protein expressed in TC-6275 revealed a 65kD moiety corresponding to the trypsin-resistant core of the Cry1F delta-endotoxin. The amino acid sequences 1 to 605 of the maize-expressed Cry1F were found to be identical to those of the native delta-endotoxin, except for a single amino acid substitution. These sequences represent the portion of the full length (1174 amino acids) Cry1F protein that remains in the insect gut after digestion by proteases.

The Cry1F protein used in toxicity studies, such as those conducted on non-target organisms, was produced microbially using Pseudomonas fluorescens into which the modified cry1F gene had been inserted. Cry1F expressed in P. fluorescens was demonstrated to be biochemically equivalent to the Cry1F expressed in TC-6275.

The PAT protein was detected and characterized in the same samples of TC-6275 that were tested for the expression of Cry1F. The mean levels of PAT in leaves were: 323 ng/mg at V9, 674 ng/mg at R1, 682 ng/mg at R4, and 0 ng/mg at senescence; in roots: 112 ng/mg at V9, 253 ng/mg at R1, 223 ng/mg at R4, and 41 ng/mg at senescence; in stalks, 282 ng/mg at R1; and in whole plants: 5 ng/mg at V9, 72 ng/mg at R1 and 18 ng/mg at senescence. Levels in forage harvested at R4 were 7 ng/mg. Grain, harvested at physiological maturity, contained 23 ng/mg. The levels of PAT in pollen were below the level of quantification.

Environmental Safety Considerations Expand

Field Testing

TC-6275 was field tested at several locations, from 1999 to 2003, in the United States and Puerto Rico. These field tests were conducted to evaluate the agronomic performance of TC-6275 (in 2002 only), the efficacy of the insecticidal protein against the targeted lepidopteran pests, and susceptibility to plant diseases (e.g. , Northern Corn Leaf Blight, smut) and non-lepidopteran insect pests (e.g. , thrips, aphids, spider mites).

Agronomic parameters that were evaluated included seed germination, emergence vigour and stand density, plant height, accumulated growing degree days to 50% pollen shed and silking, percent stalk and root lodging, percent moisture at harvest, grain yield and bushel weight (grain density). Grain yield in TC-6275 was significantly higher, compared to the near-isogenic control hybrid, at locations where the infestations of ECB were higher. However, there were generally no statistically significant differences between TC-6275 and the control hybrid for most of the agronomic parameters. While there were some statistically significant differences observed for characteristics, such as emergence vigour, stand counts, and maturity, these were not consistent and could be attributed to the effect of location rather than genotype. Seed germination in TC-6275 was not significantly different than the control hybrid and was comparable to other maize hybrids.

Results of field monitoring for susceptibility to plant diseases and non-lepidopteran insect pests revealed no differences between TC-6275 and the control hybrid. Specifically, there were no observed differences in the severity of disease symptoms and, in non-lepidopteran insect damage.

Outcrossing

Since pollen production and viability were unchanged by the genetic modification resulting in TC-6275, pollen dispersal by wind and outcropping frequency should be no different than for other maize varieties. Gene exchange between TC-6275 maize and other cultivated maize varieties will be similar to that which occurs naturally between cultivated maize varieties at the present time. In the United States, where there are no plant species closely-related to maize in the wild, the risk of gene flow to other species appears remote. Feral species related to corn, as found within the United States, cannot be pollinated due to differences in chromosome number, phenology (periodicity or timing of events within an organism’s life cycle as related to temperature and photoperiod, e.g. , flowering time) and habitat.
Maize (Zea mays ssp. mays) freely hybridizes with annual teosinte (Zea mays ssp. mexicana) when in close proximity. These wild maize relatives are native to Central America and are not present in the United States, except for special plantings. Tripsacum, another genus related to Zea, contains sixteen species, of which twelve are native to Mexico and Guatemala. Three species of Tripsacum have been reported in the continental United States: T. dactyloides, T. floridanum and T. lanceolatum. Of these, T. dactyloides, Eastern Gama Grass, is the only species of widespread occurrence and of any agricultural importance. It is commonly grown as a forage grass and has been the subject of some agronomic improvement (i.e., selection and classical breeding). T. floridanum is known from southern Florida and T. lanceolatum is present in the Mule Mountains of Arizona and possibly southern New Mexico. Even though some Tripsacum species occur in areas where maize is cultivated, gene introgression from maize under natural conditions is highly unlikely, if not impossible. Hybrids of Tripsacum species with Zea mays are difficult to obtain outside of the controlled conditions of laboratory and greenhouse. Seed obtained from such crosses are often sterile or progeny have greatly reduced fertility.

Weediness Potential
No competitive advantage was conferred to TC-6275 that would render maize weedy or invasive of natural habitats, since none of the reproductive or growth characteristics were modified. Cultivated maize is unlikely to establish in non-cropped habitats and there have been no reports of maize surviving as a weed. Zea mays is not invasive and is a weak competitor with very limited seed dispersal.

Volunteers can occur in fields, the year following cultivation, when maize is grown in rotation with other crops. Although maize volunteers are a minor weed problem, these can cause harvesting problems. TC-6275 maize volunteers will be tolerant to glufosinate ammonium, thereby compromising the use of this herbicide in glufosinate-tolerant crops (e.g., glufosinate-tolerant soybeans). However, volunteer plants may be controlled by mechanical means or by the use of other herbicides registered for use on maize volunteers.

Secondary and Non-Target Adverse Effects

The history of use and literature suggest that bacterial Bt proteins are not toxic to humans, other vertebrates, and beneficial insects. The insecticidal active core of the Bt protein (Cry1F) expressed in TC-6275 was shown to be nearly equivalent to the bacterial protein, except for one amino acid substitution.

The impact of TC-6275 maize on non-lepidopteran pests, and on non-target organisms was investigated. In field trials conducted to evaluate the efficacy of TC-6275 against the targeted lepidopteran pests of maize, plots were also monitored for damage caused by non-lepidopteran pests, such as thrips, aphids and red spider mites. There were no observed differences between TC-6275 and the non-transgenic control line in the extent of damage caused by non-lepidopteran pests.

The Cry1F protein expressed in TC-6275 is similar to that expressed by the maize line TC1507, which received regulatory approval for environmental release in the U.S. (2001), Canada (2002), and Japan (2002). This was substantiated by data, provided by Dow Agro Sciences, to demonstrate the equivalency in terms of biochemical properties and biological activity, of Cry1F in TC-6275 to that in TC1507. The following data and information on the effects of Cry1F on non-target organisms are those that were provided in support of the environmental safety of TC1507.

Maize inbreds and hybrids expressing the Cry1F protein were compared to their non-transformed counterpart for relative abundance of beneficial arthropods, including: lady beetles (Cycloneda munda and Coleomegilla maculata), predacious Carabids, brown lacewings (Hemerobiidae), green lacewings (Chrysoperla plorabunda), minute pirate bugs (Orius insidiosus), assassins bugs (Reduviidae), damsel bugs (Nabidae), Ichneumonid and Braconids (parasitic wasps), damselflies, dragonflies, and spiders. Visual counts showed no significant differences between the number of arthropods collected in the Bt maize plots and those cultivated to the non-transgenic isolines with two exceptions. There was a significantly greater number of lady beetles in the Bt maize plots (1.2 per test plant vs 0.6 per control plant), as well as significantly more Orius than in the non-transgenic control plots on two of the three sample dates. These field studies demonstrated that Cry1F had neither a direct, nor an indirect effect on beneficial arthropod populations.

Species representative of non-target and beneficial organisms in an agricultural environment were selected for acute dietary toxicity studies. The species were: honey bee larvae (Apis melifera), predatory ladybird beetle (Hippodamia convergens), green lacewing (Chysoperla carnea), parasitic Hymenoptera (Nasonia vitripennis), as well as the soil arthropod Collembola (Folsomia candida). Acute toxicity studies were also conducted with earthworms, the freshwater invertebrate Daphnia magna, Northern bobwhite quail, and mice. In all cases there were no observable adverse effects attributed to the Cry1F insecticidal protein.

An additional study was conducted on the effect of Cry1F on neonate monarch butterfly larvae when fed a 10000 ng/mL diet dose. First instar larval weight and mortality were recorded after seven days of feeding. Although there was some growth inhibition, there was no mortality to monarchs fed the 10000 ng/mL diet, the highest rate tested. Since pollen doses equivalent to 10000 ng/mL are not likely to occur on milkweed leaves in nature, it can be concluded that Cry1F will not pose a risk to monarchs.

The levels of Cry1F in TC-6275 pollen and grain were substantially lower than in TC1507 (83% less in pollen and 50% less in grain). Non-target organisms consuming TC-6275 pollen and grain would be exposed to considerably less Cry1F than from TC1507. However, soil exposure to Cry1F from TC-6275 was determined to be approximately 1.5-fold greater that from TC1507. The exposure to Cry1F in soil would nevertheless be minimal since the LC50 for Cry1F for Collembola and earthworms was at least 40-fold the estimated soil concentration from ploughed-under TC-6275 senescent plants.

Impact on Biodiversity

TC-6275 has no novel phenotypic characteristics that would extend its use beyond the current geographic range of maize production. Since the risk of outcrossing with wild relatives in the United States is remote, it was determined that risk of transferring genetic traits from TC-6275 maize to species in unmanaged environments was insignificant.

Other Considerations

In order to prolong the effectiveness of plant-expressed Bt toxins, and the microbial spray formulations of these same toxins, regulatory authorities in the United States have required developers to implement specific Insect Resistant Management (IRM) Programs. These programs are mandatory for all transgenic Bt-expressing plants, including TC-6275 maize, and require that growers plant a certain percentage of their acreage to non-transgenic varieties in order to reduce the potential for selecting Bt-resistant insect populations. Details on the specific design and requirements of individual IRM programs are published by the relevant regulatory authority.

Food and/or Feed Safety Considerations Expand

Dietary Exposure

Humans consume relatively little whole kernel or processed maize, compared to maize-based food ingredients. Maize is a raw material for the manufacture of starch, the majority of which is converted to a variety of sweetener and fermentation products, including high fructose syrup and ethanol. Maize oil is commercially processed from the germ. These materials are components of many foods including bakery and dairy goods, and the human food uses of grain from TC-6275 are not expected to be different from the uses of non-transgenic field maize varieties. As such, the dietary exposure to humans of grain from insect resistant hybrids, such as TC-6275, will not be different from that for other commercially available field maize varieties.

Nutritional Data

Forage and grain from TC-6275 maize were analyzed for nutritional composition and compared to the nutritional composition of the non-transgenic control (the near-isoline CHPH09B/2MW). In forage, harvested at the dough (R4) stage, there were no significant differences in the mean levels of protein, acid detergent fibre (ADF), neutral detergent fibre (NDF), and phosphorus between TC-6275 and the non-transgenic control line. The mean level of fat in TC-6275 was lower than the control line, but was within the range of values reported in the scientific literature. Conversely, the mean levels of ash, carbohydrate and calcium in TC-6275 were greater than those of the control line, but were also within the range of values reported in the literature.

Grain from TC-6275 and the control line were analyzed for levels of proximates (fat, protein, crude fibre, ADF, NDF, ash and carbohydrates), fatty acids, amino acids, minerals, and vitamins A, B1, B2 and folic acid. TC-6275 was found to have lower levels of crude fibre, ADF, linoleic acid, lysine, calcium, zinc, vitamin B1 and folic acid, while levels of oleic acid, isoleucine, arginine, glutamic acid, praline, iron, vitamin A and E were higher compared to the control line. Although differences were noted, these values remained within the normal range of variation reported for maize grain, with the exception of vitamin A. The levels of vitamin A were higher than the range of values reported in the literature; however, this discrepancy was attributed to different assay methods, rather than genotype.

The levels of the antinutritional compounds phytic acid and trypsin inhibitor were analyzed in TC-6275 and the control line. Statistically significant differences in the levels of these compounds were observed: levels of phytic acid were greater, and those of trypsin inhibitor were less compared to the control line. However, theses differences were within the range reported in the literature for conventional maize lines.

Toxicity and Allergenicity

The Cry1F protein expressed in TC-6275 is similar, in biochemical properties and biological activity to that expressed by the maize line TC1507. This was substantiated by data, provided by Dow Agro Sciences, to demonstrate the equivalency in terms of biochemical properties and biological activity, of Cry1F in TC-6275 to that in TC1507. The following are results from investigations on the potential for toxicity and allergenicity of Cry1F expressed maize in TC1507.

The amino acid sequence of Cry1F was compared to the sequences of known toxins and allergens using public protein sequence databases. The amino acid sequence of Cry1F was not homologous with any known toxin or allergen. Results from in vitro digestibility studies using simulated gastric fluids demonstrated rapid digestion of Cry1F: 98% of the protein was degraded within half a minute, and complete digestion was observed in less than 5 minutes. Cry1F is heat labile, as demonstrated by the loss of activity to neonate tobacco budworm after 30 minutes at 75C. The mammalian toxicity of the Cry1F protein was evaluated from results of an acute oral toxicity study. Mice were fed a dose of Cry1F (576 mg/kg body weight) which was well above the highest measured expression level in TC6275 grain (i.e. , 1.68 mg/kg). No toxic effects were observed in the mice when fed at this dose, which was estimated to be 26,000-fold greater to what humans would be exposed based on the typical consumption of foods derived from TC-6275 maize grain.

The PAT protein in TC-6275 was also evaluated for its potential for toxicity and allergenicity. An amino acid sequence homology search no similarity to known toxins and allergens. Results of previous in vitro digestibility studies on the PAT protein demonstrated its rapid digestibility in simulated gastric fluids. These results, and those of other studies previously submitted to regulatory authorities in support of the safety of PAT, substantiate its low potential for toxicity and allergenicity.

Abstract Collapse

Maize, or corn (Zea mays L.) is grown commercially in over 100 countries with a combined harvest of nearly 700 million metric tonnes in 2006. The top five producers of maize in 2005 were the United States, China, Brazil, Argentina, and Mexico, accounting for 70% of world production. Maize is grown primarily for its kernel (grain), the majority of which is used for animal feed, but with significant amounts refined into products used in a wide range of food, medical, and industrial goods.

Maize is a raw material for the manufacture of starch, the majority of which is converted by a complex refining process into sweeteners, syrups, and fermentation products, including ethanol. Maize oil is extracted from the germ of the maize kernel. Only a small proportion of the whole kernel is consumed by humans, while refined maize products such as sweeteners, starch, and oil are abundant in processed foods (e.g., breakfast cereals, dairy goods, chewing gum).

In the United States maize is typically used as animal feed, with roughly 70% of the crop fed to livestock, however a growing amount is now being used for the production of ethanol. The entire maize plant, the kernels, and several refined products such as glutens and steep liquor, are used in animal feeds. Silage made from the whole maize plant makes up 10-12% of the annual corn acreage, and is a major ruminant feedstuff. Livestock that feed on maize include cattle, pigs, poultry, sheep, goats, fish and companion animals.

Industrial uses for maize products include recycled paper, paints, cosmetics, car parts. Refined maize products are also used in bioproducts such as antibiotics.

The European corn borer (ECB), Ostrinia nubilalis, is the most damaging insect pest of maize in the United States with losses from ECB damage and control costs exceeding $1 billion each year. An average of one ECB cavity per maize stalk across an entire field can reduce yield by as much as 5% when caused by first generation larvae, and 2.5% when caused by second generation larvae, with annual yield losses estimated at 5 to 10 %. Despite consistent losses to ECB, chemical insecticides are utilized on a relatively small acreage (less than 20%). Historically, this reluctance stems from the difficulties in identifying and managing ECB in maize crops: ECB larval damage is hidden, heavy infestations are unpredictable, insecticides are costly, timing of insecticide application is difficult and multiple applications may be required to guarantee ECB control.

Weeds are also a major production problem in maize cultivation. Even a light infestation of weeds can reduce yields by 10 to 15%; severe infestations can reduce yields by 50% or more. Typically, weeds are managed using a combination of cultural (e.g., seed bed preparation, clean seed, variety selection) and chemical controls. Depending on the production area and the prevalent weed species, herbicides may be incorporated into the soil before planting (pre-plant), applied after planting but before emergence (pre-emergence), or applied after the maize plants emerge (post-emergence). Ideally, for maize production, weeds should be controlled for the full season. However, the most critical period for weed control is usually about six to eight weeks after crop emergence, during the 4th to 10th leaf stages. This critical period in the life cycle of maize must be kept weed free in order to prevent yield loss.

The transgenic maize line DAS-06275-8 (hereafter referred to as TC-6275) was genetically engineered to resist ECB, Southwestern corn borer (SWCB), fall armyworm (FAW), and black cutworm (BCW, and to a limited extent, corn earworm (CEW), by producing its own insecticide. TC-6275 was also developed to express tolerance to the herbicide glufosinate ammonium. Two novel genes, a truncated cry1F gene and the bar gene were introduced into the maize hybrid line Hi-II using Agrobacterium-mediated transformation.

The cry1F gene, isolated from the common soil bacterium Bacillus thuringiensis (Bt) var. aizawai, produces the insect control protein Cry1F, a delta-endotoxin. Cry proteins, of which Cry1F is only one, act by selectively binding to specific sites localized on the lining of the midgut of susceptible insect species. Following binding, pores are formed that disrupt midgut ion flow, causing gut paralysis and eventual death due to bacterial sepsis. Cry1F is lethal only when eaten by the larvae of lepidopteran insects (moths and butterflies), and its specificity of action is directly attributable to the presence of specific binding sites in the target insects. There are no binding sites for the delta-endotoxins of B. thuringiensis on the surface of mammalian intestinal cells, therefore, livestock animals and humans are not susceptible to these proteins.

TC-6275 maize also was developed to allow for the use of glufosinate ammonium as a weed control option, and as a breeding tool for selecting plants containing the cry1F gene. The herbicidal mode of action of glufosinate ammonium is related to the activity of glutamine synthetase (GS), the enzyme required for the synthesis of the amino acid glutamine. L-phosphinothricin, the active ingredient of glufosinate ammonium, is a structural analog of glutamate, and acts as a competitive inhibitor. After application of the herbicide, L-phosphinothricin competes with glutamine for its active sites on GS. The results of the inhibition of GS are an accumulation of ammonia in the plant, a reduction in the synthesis of glutamine, and an inhibition of photosynthesis. This causes the death of plant cells, and eventually, the entire plant. TC-6275 maize contains the bar gene, which codes for the production of the enzyme phosphinothricin acetyl-transferase (PAT). This enzyme acetylates L-phosphinothricin rendering it inactive in the plant. The PAT enzyme is not known to have any toxic properties. The bar gene was isolated from the soil bacterium Streptomyces hygroscopius, the same organism from which L-phosphinothricin was originally isolated.

TC-6275 was tested in field trials in the United States and Puerto Rico from 1999 to 2003. Data collected from these trials demonstrated that TC-6275 was not different from conventional maize varieties. TC-6275 grew normally and exhibited the expected morphology, reproductive and physiological characteristics of maize. TC-6275 was also shown not to have unexpected pest or disease susceptibility compared to conventional maize.

Maize does not have any closely related species growing in the wild in continental United States. Cultivated maize can naturally cross with annual teosinte (Zea mays ssp. mexicana) when grown in close proximity, however, these wild maize relatives are native to Central America and are not naturalized in the United States. Gene exchange between TC-6275 and maize relatives was determined to be negligible in managed ecosystems, with no potential for transfer to wild species in the United States.

TC-6275 maize was compared to its non-transgenic counterpart, a near-isogenic maize line, for the relative abundance of beneficial arthropods, such as ladybird beetles, minute pirate bugs and ichneumonids. Field studies demonstrated that Cry1F expressed in TC-6275 had neither a direct nor an indirect effect on the beneficial arthropod populations. In summary, it was determined that when compared with currently commercialized maize varieties, TC-6275 maize did not present an increased risk to or impact on interacting organisms, including humans, with the exception of specific lepidopteran insect species.

Regulatory authorities in the United States have mandatory requirements for developers of Bt maize to implement specific Insect Resistant Management (IRM) Programs. The potential exists for Bt-resistant ECB populations to develop as acreages planted with transgenic Bt hybrids expand. Hence, these IRM programs are designed to reduce this potential and prolong the effectiveness of plant-expressed Bt toxins, and the microbial Bt spray formulations that contain these same toxins.

The food and livestock feed safety of TC-6275 maize was established based on several standard criteria. As part of the safety assessment, the nutritional composition of TC-6275 grain was found to be equivalent to conventional maize as shown by the analyses of key nutrients including proximates (e.g. , protein, fat, fibre, ash and carbohydrate), amino acid composition, fatty acid profiles, minerals, and vitamins. Similar compositional analyses were conducted on TC-6275 forage, which was also found to be compositionally equivalent to forage from conventional maize.

The low potential for toxicity of plant-expressed Cry1F protein was demonstrated by the lack of amino acid sequence homology with known protein toxins, by in vitro studies showing that the protein was rapidly degraded in simulated gastric fluids, and from results of acute oral toxicity studies demonstrating no acute toxicity when Cry1F protein was fed to laboratory mice. In the latter study, mice were fed high doses of Cry1F protein with no negative consequences. The doses of Cry1F were 26,000-fold greater than levels to which humans would be exposed, based on the typical consumption of foods derived from TC-6275 maize grain.

The potential allergenicity of Cry1F was assessed by examining: physiochemical characteristics; amino acid sequence homology to known protein allergens; and digestibility in simulated gastric fluids. The Cry1F protein has a history of safe use, demonstrated by its use in microbial Bt spray formulations in agriculture and forestry for more than 30 years with no evidence of adverse effects. This fact, combined with the lack of amino acid sequence homology between Cry1F protein and known allergens, and the rapid degradation of Cry1F protein in simulated gastric fluids, were sufficient to provide with reasonable certainty that Cry1F has no allergenic potential.

The PAT protein in TC-6275 was also evaluated for its potential for toxicity and allergenicity. A sequence homology search revealed no similarity to known toxins and allergens. Results of previous in vitro digestibility studies on PAT protein reveal that it is rapidly digested in simulated gastric fluid. These results, and those of other studies previously submitted to regulatory authorities in support of the safety of PAT, demonstrate its low potential for toxicity and allergenicity.

Links to Further Information Expand


This record was last modified on Monday, March 9, 2015