Novel Food Information - Insect Protected Soybean MON 87701

Health Canada has notified Monsanto Canada Inc. that it has no objection to the food use of Insect Protected Soybean MON 87701. The Department conducted a comprehensive assessment of this variety according to its Guidelines for the Safety Assessment of Novel Foods. These Guidelines are based upon internationally accepted principles for establishing the safety of foods with novel traits.

Background

The following provides a summary of the notification from Monsanto Canada Inc. and the evaluation by Heath Canada and contains no confidential business information.

1. Introduction

Monsanto has developed Insect Protected Soybean MON 87701 using recombinant DNA techniques to introduce the cry1Ac coding sequence and its associated regulatory sequences derived from the common soil bacterium Bacillus thuringiensis subsp. kurstaki into the Glycine max (L.) Merr. genome to produce the Cry1Ac protein. Cry1Ac is an insecticidal protein with specific activity against certain lepidopteran pests.

The safety assessment performed by Food Directorate evaluators was conducted according to Health Canada's Guidelines for the Safety Assessment of Novel Foods. These Guidelines are based on harmonization efforts with other regulatory authorities and reflect international guidance documents in this area (e.g., Codex Alimentarius). The assessment considered: how MON 87701 was developed; how the composition and nutritional quality of MON 87701 compared to non-modified varieties; and the potential for MON 87701 soybeans to be toxic or cause allergic reactions. Monsanto Canada Inc. has provided data that demonstrates that MON 87701 soybeans are as safe and of the same nutritional quality as traditional soybean varieties used as food in Canada.

The Food Directorate has a legislated responsibility for pre-market assessment of novel foods and novel food ingredients as detailed in the Food and Drug Regulations (Division 28). Food use of Insect Protected Soybean MON 87701 is considered a novel food under the following part of the definition of novel foods:

"c) a food that is derived from a plant, animal or microorganism that has been genetically modified such that

1. the plant, animal or microorganism exhibits characteristics that were not previously observed in that plant, animal or microorganism."

2. Development of the Modified Plant

The petitioner has provided information describing the methods used to develop Insect Protected Soybean MON 87701 and the molecular biology data that characterize the genetic change, which confers a resistance to certain lepidopteran pests. This resistance was achieved by transformation of the conventional soybean variety A5447 with a transgenic expression cassette containing the novel gene cry1Ac (Cry1Ac protein) and its associated regulatory elements.

Insect Protected Soybean MON 87701 was genetically modified using Agrobacterium-mediated transformation of commercial soybean variety A5447 with the 2T-DNA transformation vector PV-GMIR9. The transformation vector PV-GMIR9 carried two separate transfer DNA (T-DNA I and T-DNA II) sequences, each comprised of a single gene expression cassette, one containing the codon-optimized cry1Ac coding sequence (T-DNA I) and one containing the cp4 epsps coding sequence (T-DNA II).

The cry1Ac expression cassette contains the following genetic elements: the promoter, leader, and 5' non-translated region of the Arabidopsis thaliana RbcS4 gene encoding ribulose 1,5-bisphosphate carboxylase small subunit 1A; the targeting sequence encoding the transit peptide of A. thaliana RbcS4 gene encoding small subunit 1A transit peptide to direct the Cry1Ac protein to the chloroplast; the codon-optimized cry1Ac coding sequence from B. thuringiensis subsp. kurstaki; and the 3' region of the SphasI gene of G. max encoding the 7S a' seed storage protein, ß-conglycinin, including 35 nucleotides of the carboxy terminal ß-conglycinin coding sequence with the termination codon and the polyadenylation sequence.

The cp4 epsps expression cassette contained the promoter of the 35S RNA from figwort mosaic virus (FMV), the 5' non-translated leader sequence from the A. thaliana ShkG gene encoding EPSPS, the targeting sequence encoding the chloroplast transit peptide from the ShkG gene of A. thaliana encoding EPSPS, the codon-optimized cp4 epsps coding sequence of the aroA gene from Agrobacterium sp. strain CP4 encoding the CP4 EPSPS protein, and the 3' non-translated sequence from RbcS2 gene of Pisum sativum encoding the Rubisco small subunit, which functions to direct polyadenylation of the mRNA. The T-DNA II transfer DNA containing the cp4 epsps expression cassette was inserted into the plant genome as a selective marker of transformation. The phenotype conferred by the expression of the CP4 EPSPS protein (i.e., tolerance to glyphosate-based herbicides) allowed for the selection of successful transformations. Using traditional methods of breeding, the T-DNA II transfer DNA was subsequently removed from the soybean genome, producing the MON 87701 genetic event containing only the cry1Ac expression cassette (T-DNA I).

The Cry1Ac protein is a crystal delta-endotoxin from B. thuringiensis subsp. kurstaki that binds to specific cadherin receptors on the midgut epithelium of targeted lepidopteran insects. Once bound, the endotoxin forms cation selective pores, which leads to the inhibition of the insect's digestive processes.

3. Characterization of the Modified Plant

Southern blot analysis and DNA sequencing of Insect Protected Soybean MON 87701 demonstrated the presence of a single copy of the cry1Ac expression cassette in the soybean genome at a single locus. Southern blot analysis confirmed the absence of any plasmid backbone DNA in Insect Protected Soybean MON 87701.

The stability of the inserted cry1Ac expression cassette was evaluated from the progeny of five different generations. The results of Southern blot analysis and segregation data demonstrated the stability of Insect Protected Soybean MON 87701 at the genomic level.

4. Product Information

Insect Protected Soybean MON 87701 differs from its traditional counterpart by the addition of the cry1Ac gene sequence and its associated regulatory elements. The insertion of this gene results in the tissue-targeted expression of the novel Cry1Ac protein in MON 87701. The expression of Cry1Ac confers resistance to certain lepidopteran pests.

Cry proteins have a history of safe use including their use in bacterial preparations used as biopesticides. The U.S. Environmental Protection Agency (EPA) has established separate tolerance exemptions for various Cry proteins (e.g. Cry1Ab, Cry1Ac, Cry2Ab2, Cry3Aa, and Cry3Bb1). Monsanto's Bollgard®^{Footnote 1} cotton also expresses the Cry1Ac protein and has been used commercially in the U.S. for over ten years. A related protein, Cry1Ab, which shares 90% amino acid identity to the Cry1Ac protein expressed in Bollgard® cotton and MON 87701, is expressed in YieldGard®^{Footnote 2} Corn Borer corn. An exemption from the requirement for a tolerance was granted in 1996 for Cry1Ab and the genetic material necessary for its production in all plants (40 CFR § 174.511). A tolerance exemption for the Cry1Ac protein and the genetic material necessary for its production in all plants was granted on April 11, 1997 (40 CFR § 174.510). The safety for food and feed consumption of plants expressing Cry1Ac or Cry1Ab proteins has been evaluated by several regulatory agencies around the world including the U.S. FDA.

The petitioner has provided data to demonstrate the level of expression of the Cry1Ac protein in MON 87701. This study used plant samples from five field trial locations conducted in the United States: Alabama, Arkansas, Georgia, Illinois, and North Carolina. All locations are relevant soybean-growing regions and represent a range of environmental conditions typically encountered in the production of soybean. At each field site, three replicated plots of MON 87701 and a conventional soybean control (A5547) were planted using a randomized complete block field design. Tissues of MON 87701 that were collected include: Over-season leaf (OSL), forage, root, harvested seed, and pollen. The quantities of Cry1Ac protein were determined by an enzyme-linked immunosorbent assay (ELISA). Protein quantities for the tissues were calculated on a microgram (mg) per gram (g) dry weight (dwt) basis. The level of Cry1Ac was below the limit of detection (LOD) for all tissue types of A5547. For MON 87701, Cry1Ac levels were detected at the highest levels in OSL-4 leaf tissue (340 ± 290 mg/g dwt), followed by forage (34 ± 36 mg/g dwt), and mature, harvested seed (4.7 ± 0.79 mg/g dwt).

5. Dietary Exposure

Insect Protected Soybean MON 87701 is expected for use in similar applications as other soybean and vegetable oils by the food industry. The petitioner has indicated that the MON 87701 product concept is to reduce or replace current insecticide applications to control lepidopteran pests of soybean in tropical and subtropical production regions, where these insects cause significant plant damage and yield loss. The petitioner is not aware of food or feed uses of conventional soybean that are not applicable to MON 87701.

6. Nutrition

The nutrient data pertaining to the MON 87701 and the control variety A5547 were obtained from trials conducted at five locations conducted during the 2007 growing season. A randomized complete block design was used with three replicates per block. Commercial conventional soybean varieties were also grown to establish a reference range for different analytes.

The key nutritional parameters measured in soybean seed were: proximates (ash, fat, moisture, and protein), amino acids (arginine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine), fatty acids (palmitic, stearic, oleic, linoleic, linolenic and arachidic), phytic acid, trypsin inhibitors, lectin, and isoflavones (daidzein, glycitein, and genestein).

The nutritional composition of soybeans from transgenic and non-transgenic samples was comparable. In the combined site analysis, there were 12 analytes that showed differences in composition between MON 87701 and the control. These differences were not consistently observed across sites, and all values were within the established reference range as well as comparable to literature (ILSI) values.

7. Chemistry/Toxicology

The Insect Protected Soybean MON 87701-produced Cry1Ac protein is derived from the non-pathogenic B. thuringiensis subsp. kurstaki soil microorganism that is abundant in the environment and has a history of safe use for pest control in agriculture.

Acute toxicity tests showed no treatment-related adverse effects of mice were observed when the Cry1Ac protein was administered as a single dose of up to 1460 mg/kg BW (80% purity).

A dietary safety assessment shows that the margin of exposure (MOE; MOE = NOEL (mg/kg/ Exposure (mg/kg/day)) for the overall U.S. population is =2.93 ´ 106. This large value indicates there is a very low risk associated with human exposure to the Cry1Ac protein.

Digestive fate experiments demonstrated that the full length Cry1Ac protein is rapidly digested in simulated gastric fluid (SGF), apart from the small transiently stable fragment that was quickly degraded during a short exposure to simulated intestinal fluid (SIF). This indicates that it is highly unlikely that the Cry1Ac protein will reach absorptive cells of the intestinal mucosa, resulting in low to no allergenic risk.

The levels of endogenous allergens in Insect Protected Soybean MON 87701 are comparable to those in soybean varieties that are currently available on the market.

Conclusion

Health Canada's review of the information presented in support of the food use of Insect Protected Soybean MON 87701 does not raise concerns related to food safety. Health Canada is of the opinion that food derived from MON 87701 soybeans is as safe and nutritious as food from current commercial soybean varieties.

Health Canada's opinion deals only with the food use of Insect Protected Soybean MON 87701. Issues related to its use as animal feed have been addressed separately through existing regulatory processes in the Canadian Food Inspection Agency (CFIA). The CFIA evaluated information provided on the environmental, animal, and human health safety of Insect Protected Soybean MON 87701 with the intended use in animal feed. From their assessment, the CFIA concluded that there are no concerns from an environmental and feed safety perspective. This perspective is applicable to the food and feed products derived from Insect Protected Soybean MON 87701 destined for commercial sale.

It is the continuing responsibility of the food manufacturer or importer to ensure that their products are in compliance with all applicable statutory and regulatory requirements. Any new information obtained in relation to these products which have potential health and safety implications should be forwarded to Health Canada for our consideration in order to ensure the continued safety and integrity of all foods available in the Canadian marketplace. The sale of a food which poses a hazard to the health of consumers would contravene the provisions of the Food and Drugs Act.

This Novel Food Information document has been prepared to summarize the opinion regarding the subject product provided by the Food Directorate, Health Products and Food Branch, Health Canada. This opinion is based upon the comprehensive review of information submitted by the petitioner according to the Guidelines for the Safety Assessment of Novel Foods.

(Également disponible en français)

For further information, please contact:

Novel Foods Section
Food Directorate
Health Products and Food Branch
Health Canada, PL2204A1
251 Frederick Banting Driveway
Ottawa, Ontario K1A 0K9
novelfoods-alimentsnouveaux@hc-sc.gc.ca