Novel Food Information - Herbicide Tolerant Cotton Event GHB811

Health Canada has notified BASF Canada Inc. that it has no objection to the food use of herbicide tolerant cotton event GHB811 (GHB811). This event was initially submitted to the Department as an asset owned by Bayer CropScience Inc. During the assessment of this event, the ownership of this asset was transferred to BASF Canada Inc. The Department conducted a comprehensive assessment of this cotton 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 original notification from Bayer CropScience Inc. and the evaluation by Health Canada and contains no confidential business information.

1. Introduction

Bayer CropScience Inc. has developed a genetically modified Gossypium hirsutum (cotton) variety which exhibits tolerance to glyphosate and HPPD inhibitor herbicides.

GHB811 was developed through the introduction of two genes: a 2mepsps gene and an hppdPfW336-1Pa gene, encoding a mutated 5-enol pyruvylshikimate-3-phosphate synthase (2mEPSPS) and a mutated 4-hydroxyphenylpyruvate dioxygenase (HPPD W336), respectively. The 2mepsps gene has been previously assessed by Health Canada in herbicide tolerant soybean event FG72 and GyTol™ cotton event GHB614 (2012 and 2008, respectively). The hppdPfW336-1Pa gene has been previously assessed by Health Canada in herbicide tolerant soybean event FG72 (2012).

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 this cotton variety was developed; how the composition and nutritional quality of this variety compared to non-modified cotton varieties; and the potential for this cotton variety to be toxic or cause allergic reactions. Bayer CropScience Inc. has provided data that demonstrate that GHB811 is as safe and of the same nutritional quality as traditional cotton 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 B.28). GHB811 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:

• 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 GHB811 and the molecular biology data that characterize the genetic change, which results in tolerance to glyphosate and HPPD inhibitor herbicides.

GHB811 was developed through Agrobacterium-mediated transformation of cotton variety Coker 312 with a transformation vector (pTSIH09) containing a construct (T-DNA) of an 2mepsps gene and a hppdPfW336-1Pa gene, encoding a mutated 5-enol pyruvylshikimate-3-phosphate synthase (2mEPSPS) and a mutated 4-hydroxyphenylpyruvate dioxygenase (HPPD W336), respectively.

The coding sequence for the 2mEPSPS was isolated from maize (Zea mays L.). Two amino acids were substituted (threonine by isoleucine at position 102, I102T; and proline by serine at position 106, S106P). These modifications confer to the protein a decreased binding affinity for glyphosate, allowing it to be tolerant to glyphosate and to maintain sufficient enzymatic activity in the presence of the herbicide. The modified protein is designated as 2mEPSPS. The amino acid sequence of the 2mEPSPS protein expressed in cotton event GHB811 is identical to the one expressed in double herbicide tolerant soybean event FG72 and GyTol™cotton event GHB614 (both previously assessed by Health Canada in 2012 and 2008, respectively).

The coding sequence for the HPPD W336 protein was isolated from the bacterium Pseudomonas fluorescens Migula, strain A32. One amino acid was substituted (glycine by tryptophan at position 336, W336G) to be tolerant to HPPD inhibitor herbicides and to maintain sufficient enzymatic activity. The modified protein is designated as HPPD W336. The amino acid sequence of the HPPD W336 protein expressed in cotton event GHB811 is identical to the one expressed in double herbicide tolerant soybean event FG72 (assessed by Health Canada in 2012).

The petitioner provided information to support the safety and historical use of each donor organism (Z. mays L. and P. fluorescens Migula) and the recipient organism (Gossypium hirsutum L.). None of these organisms pose a health safety concern.

3. Characterization of the Modified Plant

The number of integration sites of the T-DNA insert in GHB811 was characterized by means of Southern blot analysis on genomic DNA (gDNA) prepared from leaf material. Results of the Southern blot analysis demonstrated the presence of a single, intact copy of the T-DNA insert in the GHB811 genome. Results of the Southern blot analysis also confirmed the absence of all vector backbone sequence in the GHB811 genome.

Bioinformatics analysis on the GHB811 pre-insertion locus sequence was performed to identify the position of the pre-insertion locus in the genome and to determine whether any regulatory sequences or endogenous genes were interrupted upon insertion of the T-DNA insert. Based on the bioinformatics analysis, it was demonstrated that the pre-insertion locus is located on the cotton chromosome A05 and that it is unlikely that the insertion of the T-DNA insert in the GHB811 pre-insertion locus interrupts or alters the transcription or translational activity of endogenous cotton genes.

Bioinformatics analyses were performed on the GHB811 T-DNA insert and flanking gDNA sequences to identify potential open reading frames (ORFs). An ORF was defined as the region between two translation stop codons (TAA, TAG, or TGA) with a minimum size coding for 3 amino acids. Translated amino acid sequences from all identified ORFs with a minimum size of 30 amino acids were used as query sequences for a homology search with known allergens and toxins. In the GHB811 T-DNA insert locus sequence, 549 ORFs were identified. After elimination of duplicates, translated amino acid sequences of at least 30 amino acids represented 126 unique sequences. None of the identified ORFs show any allergenic or toxicological potential.

Genetic stability of the T-DNA insert in the GHB811 genome was demonstrated by assessing individual GHB811 plants from five generations (T1, T3, T4, BC1F2, and BC2F3) by means of Southern blot analysis. The results from the Southern blot analysis demonstrated the genetic stability of the T-DNA insert in the GHB811 genome over multiple generations. Chi-square (χ2) analysis of the segregation data for the three generations was performed to test the hypothesis that the T-DNA insert in GHB811 is inherited in a manner that is predictable according to Mendelian principles and is consistent with insertion into a single chromosomal locus within the cotton nuclear genome. The results are consistent with Mendelian principles of inheritance and support the conclusion that the GHB811 genome contains a single T-DNA insert integrated into a single chromosomal locus within the cotton nuclear genome.

4. Product Information

GHB811 differs from its traditional counterparts by the addition of two genes: 2mepsps, encoding encoding a mutated 5-enol pyruvylshikimate-3-phosphate synthase (2mEPSPS); and hppdPfW336-1Pa, encoding a mutated 4-hydroxyphenylpyruvate dioxygenase (HPPD W336), respectively.

 expression levels of 2mEPSPS and HPPD W336 were determined by enzyme-linked immunosorbent assay (ELISA) in field-grown cotton matrices from GHB811 plants treated and untreated with trait-specific herbicides grown at three field trials in the USA in 2015. Two plots of GHB811 were included at each field trial site. One plot was treated with trait-specific herbicide while the other plot was untreated. An application of isoxaflutole to the treated GHB811 plants was made at a rate of 104.9 to 106.6 g ai/ha before emergence (BBCH 00). An application of glyphosate was made at a rate of 1104 to 1123 g ai/ha at the seven to eight leaf growth stage (BBCH 17-18). All plants were of the Coker 312 background.

Leaf tissue at BBCH 60-67 and BBCH 51-55 growth stages demonstrated the highest mean 2mEPSPS protein expression levels. Mean (±SD) 2mEPSPS expression levels in untreated and treated leaf at BBCH 60-67 of GHB811 was 1422.12 ± 206.41 mg/g DW and 1267.95 ± 247.75 mg/g DW, respectively. Mean (±SD) 2mEPSPS expression levels in untreated and treated leaf at BBCH 51-55 of GHB811 was 1344.37 ± 224.96 m/g DW and 1269.39 ± mg/g DW, respectively. Fuzzy seed (BBCH 83-97) demonstrated the lowest mean 2mEPSPS protein expression in all matrices reported on a DW basis. Mean (±SD) 2mEPSPS expression levels in untreated and treated fuzzy seed of GHB811 was 145.11 ± 37.86 mg/g DW and 150.88 ± 27.87 ug/g DW, respectively. The level of 2mEPSPS protein expression in all tissue samples were similar between the GHB811 plants treated with trait-specific herbicides and untreated GHB811 plants. Thus, treatment with the trait-specific herbicides does not appear to affect 2mEPSPS protein expression in GHB811.

Leaf tissue at BBCH 51-55 growth stage demonstrated the highest mean HPPD W336 protein expression levels. Mean (±SD) HPPD W336 protein expression levels in untreated and treated GHB811 leaf at BBCH 51-55 was 1043.64 ± 322.96 ug/g DW and 956.75 ± 204.79 mg/g DW, respectively. Root (BBCH 14-16) demonstrated the lowest mean HPPD W336 protein expression levels in all matrices reported on a DW basis. Mean (±SD) HPPD W336 expression levels in untreated and treated root of GHB811 was 22.12 ± 8.37 mg/g DW and 25.42 ± 10.98 mg/g DW, respectively. Mean (±SD) HPPD W336 expression levels in untreated and treated fuzzy seed (BBCH 83-97) of GHB811 was 29.61 ± 14.96 mg/g DW and 27.01 ± 9.78 mg/g DW, respectively. The level of HPPD W336 protein expression in all tissue samples were similar between the GHB811 plants treated with trait-specific herbicides and untreated GHB811 plants. Thus, treatment with the trait-specific herbicides does not appear to affect HPPD W336 protein expression in GHB811.

5. Dietary Exposure

It is expected that GHB811 will be used in applications similar to conventional cotton varieties. The petitioner does not anticipate a significant change in the food use of cotton with the introduction of GHB811.

6. Nutrition

Eight field trials were used to collect samples for compositional analysis of GHB811 and its control, non-GM counterpart (Coker312).  The field trials were conducted in cotton growing regions of the USA in 2014 (5 sites) and 2015 (3 sites). GHB811 (untreated and treated with herbicide), and Coker312 were grown at all sites.  Each of the three lines was replicated four times in a randomized complete block design. In addition, compositional analysis was conducted on several non-GM reference varieties grown at each site. Herbicide-treated GHB811 received one application of isoxaflutole and one application of glyphosate herbicide. Typical commercial agriculture production practices were used for the field trials.

Cotton seed samples were analyzed for moisture, protein, fat, ash, total dietary fibre, neutral detergent fibre (NDF), acid detergent fibre (ADF), carbohydrate (by difference), amino acids, fatty acids, vitamin E (α-tocopherol), minerals and anti-nutrients (free and total gossypol, and malvalic acid, sterculic acid, and dihyrosterculic acid). Compositional analysis was conducted by EPL Bio Analytical Services using acceptable methods.

The petitioner analyzed a total of 54 analytes. Statistical analysis was completed using SAS version 9.3 and significant differences were noted when t-test p-values were < 0.05. The analysis of GHB811 (untreated) and control Coker 312 showed six statistically significant differences in the combined site analysis which occurred in: protein, vitamin E, palmitoleic acid (C16:1), stearic acid (C18:0), free gossypol, and total gossypol, and 11 statistically significant differences in GHB811 (treated) and control for: protein, vitamin E, palmitoleic acid (C16:1), stearic acid (C18:0), neutral detergent fibre (NDF), methionine, cysteine, arachidic acid (C20:0), free gossypol, total gossypol, and dihydrosterculic acid. Of the analytes listed above, all were lower in GHB811 except for palmitoleic acid and NDF which were higher. In all cases the differences were small, and the analyte levels were within the reference ranges provided by the petitioner for the respective analyte.

Based on the information provided on the composition of GHB811 and control Coker 312, the Nutrition Pre-market Assessment Division (NPAD) concludes that there are no safety concerns with the use of GHB811 cotton as a food ingredient in Canada from a nutritional perspective

7. Chemistry/Toxicology

The petitioner submitted new data which served to support the safety assessment of the 2mEPSPS and HPPD W336 proteins. These studies included acute oral toxicity studies conducted with the novel proteins and updated searches of the publicly available databases assessing the sequence similarity of these proteins to known toxins.

Acute oral toxicity studies conducted in mice with each of the novel proteins demonstrated that neither 2mEPSPS nor HPPD W336 resulted in any treatment-related effects. The oral LD50 for 2mEPSPS protein is greater than 2000 mg/kg bw. The oral LD50 for HPPD W336 protein was also determined to be greater than 2000 mg/kg bw. The updated bioinformatics analyses indicated that neither the 2mEPSPS nor HPPD W336 proteins demonstrated significant sequence homology with known toxic proteins.

The presence of toxins endogenous to cotton was assessed for GHB811. Gossypol and cyclopropenoid fatty acids do not pose a health concern at the levels present.

A conservative dietary exposure assessment based on the consumption of cotton seed by the United States (US) population was provided. With this information, the difference between the estimated dietary exposure and the acute oral LD50 for both 2mEPSPS and HPPD W336 protein was calculated and was considered sufficiently large in both cases (greater than 100,000), supporting the safety of GHB811.

Based on the information provided, the Pre-market Toxicology Assessment Section (PTAS) has no safety concerns regarding GHB811 from a toxicological perspective.

The PTAS considered new safety information in support of the allergenicity assessment which included an assessment of in vitro digestibility and updated searches of the publicly available databases assessing the sequence similarity of 2mEPSPS and HPPD W336 proteins to known allergens. The bioinformatics analyses indicated that neither 2mEPSPS nor HPPD W336 proteins shared significant sequence identity with known and putative allergens.

Regarding the proposed food uses of GHB811, 2mEPSPS and HPPD W336 proteins are considered not likely to provoke an allergenic response in the general population. As such, the PTAS has no safety concerns regarding GHB811 from an allergenic perspective.

Conclusion:

Health Canada’s review of the information presented in support of the food use of GHB811 does not raise concerns related to food safety. Health Canada is of the opinion that food derived from this cotton variety is as safe and nutritious as food derived from current commercial cotton varieties.

Health Canada's opinion deals only with the food use of GHB811. Issues related to its use as animal feed have been addressed separately through existing regulatory processes in the CFIA.

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.

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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