Biotechnology Consultation Note to the File BNF No. 000077

Date: September 4, 2002

Subject: Monsanto Company's Glyphosate-Tolerant Canola Line GT200. BNF 000077.

Keywords: Canola, Brassica napus, glyphosate-tolerant, herbicide tolerant, CP4 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS), glyphosate oxidoreductase (GOX), Roundup™ Ready.

Background

In a submission dated April 30, 2001, and letters dated July 6 and October 1, 2001, Monsanto Company (Monsanto) provided summary data and information supporting their safety assessment of glyphosate-tolerant canola line GT200. Although Monsanto has no intention of commercializing the GT200 line, they have initiated the consultation process because of their determination that GT200 has the potential to be present at low, adventitious levels in commercial canola varieties. Monsanto developed the GT200 line in the early 1990s along with another glyphosate-tolerant canola line, GT73. Both lines express glyphosate oxidoreductase (GOX) and CP4 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) but are derived from distinct transformation events. The GOX protein expressed in the GT73 line differs from the GOX protein expressed in the GT200 line by three amino acids. The CP4 EPSPS and GOX proteins provide tolerance to glyphosate herbicide. After field testing both lines, Monsanto selected line GT73 for commercial development and completed a consultation on that line in 1995 (BNF 000020).

Edible oil is the sole product from canola that is consumed by humans. Heat-processed canola meal is used solely as a protein-rich livestock feed and is not consumed by humans.

Molecular Alterations and Characterization of Canola Line GT200

Canola line GT200 was created by Agrobacterium tumefaciens mediated transformation of the Westar commercial canola variety using plant transformation vector PV-BNGT03. Vector PV-BNGT03 contains a plasmid backbone which is not intended to be transferred to the recipient canola line as well as a "T-DNA" region that is intended to be transferred to the recipient canola line. The T-DNA includes the following genetic elements intended to be transferred to the recipient organism:

• 35S promoter from a modified figwort mosaic virus
  
• N-terminal of the small subunit 1A of the ribulose-1,5-bisphosphate carboxylase chloroplast transit peptide from Arabidopsis
• synthetic glyphosate oxidoreductase gene (the gox gene) based on the glyphosate oxidoreductase gene isolated from Ochrobactrum anthropi strain LBAA
• 3' end of the pea rbcS E9 gene, which provides the polyadenylation sites for the gox and CP4 epsps genes
• N-terminal chloroplast transit peptide sequence from the Arabidopsis EPSPS gene
• synthetic 5-enolpyruvylshikimate-3-phosphate synthase gene based on the sequence from Agrobacterium sp. strain CP4 (the CP4 epsps gene).

The plasmid PV-BNGT03 backbone contains the following elements that are not intended to be transferred to the recipient organism:

• ori-V from plasmid RK2 which allows plasmid replication in Agrobacterium
• ori-322 which permits vector propagation in E. coli
• aad gene encoding the Tn7 AAD 3' adenylyltransferase that confers spectinomycin and streptomycin resistance to bacterial cells carrying PV-BNGT03.

Monsanto reports that molecular analysis of canola line GT200 shows that a single copy of the T-DNA from PV-BNGT03 was inserted into the genomic DNA of Westar canola. Southern blot analysis was used to demonstrate that only the sequences of the T-DNA, and not sequences from the PV-BNGT03 backbone, are present in canola line GT200. Thus, the transgenic DNA at the insertion site is composed of the CP4 epsps and gox genes and DNA sequences that direct the expression and cellular targeting of the CP4 EPSPS and GOX proteins. Inheritance data showing that the glyphosate-tolerance phenotype was inherited as a single dominant Mendelian trait confirmed the presence of a single T-DNA insert in GT200 canola.

Expressed Proteins

Monsanto measured the levels of CP4 EPSPS and GOX proteins in canola line GT200 and determined that the proteins were present in leaf tissue of canola plants homozygous for the GT200 insertion at levels of 0.031 micrograms/milligram (µg/mg) tissue (fresh weight) and 0.069 µg/mg tissue (fresh weight), respectively. The company reported that CP4 EPSPS and GOX were present in canola seeds homozygous for the insertion at levels of 0.051 µg/mg tissue (fresh weight) and 0.142 µg/mg tissue (fresh weight), respectively. Seeds heterozygous for the insertion contained 0.034 µg/mg of CP4 EPSPS and 0.105 µg/mg of GOX on a fresh weight basis. The absolute levels of the GOX and CP4 EPSPS proteins in seeds homozygous for the insert account for less than 0.07 percent of the seed on a fresh weight basis. The CP4 EPSPS and GOX proteins were not detected in the parent canola line, Westar, as expected. Given the low expression levels of CP4 EPSPS and GOX, Monsanto does not consider these proteins to be potential macroconstituents of the human or animal diet.

Monsanto searched protein databases for sequences homologous to CP4 EPSPS and GOX, and concluded that the CP4 EPSPS and GOX proteins are not meaningfully homologous to known protein allergens or known toxins. Based on studies using simulated gastric fluid and simulated intestinal fluid, Monsanto reports that the CP4 EPSPS and GOX proteins are readily digested in the mammalian digestive tract. Monsanto also states that there is no detectable protein in canola oil, the only human food product that would be derived from canola. Monsanto also reports that the heat processing that would be part of the method of manufacture of any canola meal that could be used for livestock feed would inactivate and denature proteins contained in the meal. The company also reports that no treatment-related adverse effects were observed in acute toxicity tests in which mice were gavaged with doses of up to 572 mg CP4 EPSPS/kilogram (kg) body weight or up to 100 mg GOX/kg body weight.

Compositional Analysis of Seeds

A. Nutrients

Monsanto measured proximates⁽¹⁾, amino acids⁽²⁾, and fatty acids⁽³⁾ in seed samples obtained from seven field trials with canola line GT200 and the parental line (Westar). The results of these analyses show that the levels of these nutrients in line GT200 are not materially or meaningfully different from those in the parental line Westar (the ranges for all the nutrients measured overlapped). Where literature values were available, the levels of all the nutrients measured in line GT200 fell within the published values for commercial canola varieties.

B. Natural Toxicants/Antinutrients

Monsanto evaluated canola seeds from line GT200 for the possible presence of increased levels of erucic acid, glucosinolates, and sinapine, which occur naturally in canola. Erucic acid is a potentially cardiopathic substance in experimental animals. The level of erucic acid in oil from canola line GT200 was similar to the amount in oil from the Westar control and is below the limit of 2 percent in low erucic acid rapeseed oil affirmed as generally recognized as safe (GRAS) for human consumption (21 CFR § 184.1555). Monsanto also concludes that there are no meaningful differences in the levels of glucosinolates (antinutritional factors in animal feed) between the GT200 and Westar varieties and that the level of alkyl glucosinolates in GT200 is well below the limit of 30 µmole/gram of oil-free meal established for the safe use of meal derived from canola seed as an animal feed. Sinapine, a family of choline esters in canola, is an undesirable component of poultry feed because it may cause an off-odor in chicken eggs. Based on analysis of sinapine levels of defatted meal samples, Monsanto concludes that there is no meaningful difference in the level of choline esters in the seeds of the GT200 and Westar canola varieties. The level of sinapine in these lines is also consistent with those reported in the literature for commercial varieties of canola.

Compositional Analysis of Canola Meal

Toasted canola meal is used as a feedstuff for livestock, poultry, hogs, and fish. Monsanto analyzed toasted meal for proximates⁽⁴⁾ and nitrogen solubility. The company compared one sample of the meal derived from seed of the Westar line with two samples of meal derived from mixtures of seeds from glyphosate-tolerant canola lines GT200 and GT73. Seed from lines GT200 and GT73 had been inadvertently commingled in the field. The seed commingling was discovered after the analyses were completed. The commingled seed mixtures contained approximately 50 percent GT200 seed and approximately 50 percent GT73 seed. The company concludes that the processed canola meal from the GT200 and GT73 canola seed mixture is not meaningfully different in proximate analysis from processed canola meal currently in commerce. Thus, Monsanto concludes that the composition of canola seeds and toasted meal from line GT200 are not materially different from Westar canola seeds or meal.

Animal Wholesomeness Studies

Monsanto submitted animal wholesomeness studies with glyphosate-tolerant canola in both the current consultation as well as a previous one (BNF 000020). Most of the studies submitted in this consultation were also submitted in BNF 000020 and utilized glyphosate-tolerant canola that was a mixture of canola line GT73 and line GT200. In the current consultation, Monsanto also submitted a new rat feeding study conducted in 1996 (Naylor, 1996) that used only GT73 canola. Monsanto believes that wholesomeness studies with the mixture of lines GT73 and GT200 canola are generally applicable to assessing the wholesomeness of canola from the current GT200 line as well as the GT73 line. Both GT73 and GT200 canola are engineered to express two new proteins, CP4 EPSPS and GOX, that are not present in the parental (Westar) line. Monsanto stated that the CP4 EPSPS protein is identical in the GT73 and GT200 lines, while the GOX proteins in the two lines differ by only three amino acids, making them greater than 99 percent identical. Because of the identity of the CP4 EPSPS proteins and the close similarity of the GOX proteins in GT73 and GT200 canola, Monsanto believes that wholesomeness studies with GT73 canola are generally applicable to assessing the wholesomeness of both canolas.

Summaries and results of wholesomeness studies in BNF 000020 (Ref. 184-188) included two 4-week rat studies with glyphosate-tolerant canola meal (Naylor 1994⁽⁵⁾, 1995⁽⁶⁾). In the rat studies, wholesomeness was assessed by monitoring growth, food consumption, gross pathology, liver weight, and kidney weight. In the 1994 study (which utilized meal from both GT200 and GT73 canola), organ weights (kidney, liver) were comparable in rats fed meal from parental and glyphosate-tolerant canola. However, statistically significant decreases (approximately minus 10%) in terminal body weight and cumulative body weight gains were observed in male rats, but not females, fed processed (5 or 15%) or unprocessed (15%) meal from glyphosate-tolerant canola, when compared to rats fed canola meal from the parental (Westar) line. After completion of the study, Monsanto discovered technical problems ⁽⁷⁾ with the test article. These technical problems were considered to impact on the reliability of the study findings, necessitating that the study be repeated.

In the repeat study (Naylor, 1995), rats fed processed glyphosate-tolerant canola meal had normal weight gain, but relative liver weights were increased by up to 16 percent in comparison to rats fed meal from the parental canola line. Monsanto considered these results to be inconsistent with the findings from the 1994 study, and decided to conduct a third more comprehensive wholesomeness study (Naylor, 1996⁽⁸⁾), which is summarized in the current BNF (000077).

The 1996 study utilized meal from a series of 8 separate commercial canola varieties as controls in order to provide information on the normal range of biological response to rats fed processed canola meal. The 1996 study reported no difference in growth or absolute/relative organ weight (liver, kidney) in rats fed glyphosate-tolerant canola meal (line GT73) in comparison to meal from a wide variety of canola controls. Liver weights were well within the range observed in rats fed the different varieties of commercial canola and no gross pathological changes were associated with consumption of glyphosate-tolerant canola meal. Monsanto believes that the 1996 wholesomeness study of GT73 canola in rats is more comprehensive than the two previous rat studies in BNF 000020 and is appropriate for assessing the wholesomeness of canola from GT200.

Monsanto also performed a 10-week trout study with processed canola meal and a five-day quail study with unprocessed canola meal. Both meals were derived from GT200 and GT73 seeds and incorporated into the diet at levels of up to 20%. In both studies, body weight gains were no different relative to values obtained in animals fed the Westar parental control line.

Conclusions

Monsanto has no intention of commercializing canola line GT200, but has initiated the consultation process because line GT200 has the potential to be present at low, adventitious levels in commercial canola varieties. Monsanto has concluded that glyphosate tolerant canola line GT200 is not materially different in composition, safety, wholesomeness, or any relevant parameter from canola now grown, marketed, and consumed.

At this time, based on Monsanto's description of its data and information, the Agency considers Monsanto's consultation on transgenic canola line GT200 to be complete.

 

Jason Dietz

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⁽¹⁾Proximate analysis of canola seeds included crude protein, ash, moisture, fat, crude fiber and carbohydrates (calculated by difference).

⁽²⁾Monsanto assessed the amino acid composition of the canola seed protein. Monsanto measured the following amino acids: alanine, arginine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Monsanto reported the level of each amino acid in g/100 g protein dry weight.

⁽³⁾Monsanto assessed the fatty acid composition of the oil fraction of canola seeds. Monsanto measured the following fatty acids: C16:0, C16:1, C18:0, C18:1, C18:2, C18:3, C20:0, C20:1, C20:2, C22:0, and C22:1. Monsanto reported the level of each fatty acid as a percent of total fatty acids.

⁽⁴⁾Proximate analysis of canola meal included crude protein, fat, crude fiber, ash, moisture, carbohydrate (by calculation), and energy content (by calculation).

⁽⁵⁾Lines GT200 and GT73, processed and unprocessed meal

⁽⁶⁾Line GT73, processed meal only

⁽⁷⁾The technical problems included inadvertent commingling of glyphosate-tolerant seed samples, preparation of non-representative composite seed samples, and possible differences in processing of the ground seed samples.

⁽⁸⁾ Line GT73, processed meal only