Novel Food Information: Soybean Event GMB151
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Background:
Health Canada has notified BASF Canada Inc. that it has no objection to the food use of Soybean Event GMB151 (GMB151). The Department conducted a comprehensive assessment of this soybean 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.
The following provides a summary of the notification from BASF Canada Inc. and the evaluation by Health Canada and contains no confidential business information.
1. Introduction
BASF Canada Inc. has developed a genetically modified Glycine max L. (soybean) variety which exhibits resistance to the soybean cyst nematode (SCN), Heterodera glycines ichinohe, and tolerance to 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicides.
GMB151 was developed through the introduction of two genes: a cry14Ab-1.b gene encoding a crystal (Cry14Ab-1) protein, and a hppdPf-4Pa gene encoding a 4-hydroxyphenylpyruvate dioxygenase (HPPD-4) protein.
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 soybean variety was developed; how the composition and nutritional quality of this variety compared to non-modified soybean varieties; and the potential for this soybean variety to be toxic or cause allergic reactions. BASF Canada Inc. has provided data that demonstrate that GMB151 is 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 B.28). Foods derived from GMB151 soybean are considered novel foods 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 GMB151 and molecular biology data that characterized the genetic change, which results in resistance to the soybean cyst nematode (SCN), Heterodera glycines ichinohe, and tolerance to 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicides.
GMB151 was developed through Agrobacterium-mediated transformation of soybean variety Thorne with a transformation vector pSZ8832. The pSZ8832 vector contains a construct (T-DNA) of the cry14Ab-1.b gene, encoding a crystal (Cry14Ab-1) protein, and the hppdPf-4Pa gene, encoding a 4-hydroxyphenylpyruvate dioxygenase (HPPD-4) protein.
The coding sequence for the Cry14Ab-1 protein was isolated from Bacillus thuringiensis, a Gram positive, spore-forming bacterium. To date, more than 700 cry gene sequences that code Cry proteins have been identified. Cry proteins typically have narrow target specificity that limit activity of individual proteins to just a few genera. Cry proteins have been successfully used as biopesticides against Lepidoptera (butterflies and moths), Coleoptera (beetles), and Diptera (flies). Expression of the Cry14Ab-1 protein confers resistance to the soybean cyst nematode (SCN), Heterodera glycines ichinohe.
The coding sequence for the HPPD-4 protein was isolated from Pseudomonas fluorescens strain A32, a Gram negative, rod-shaped, motile, asporogenous, aerobic bacterium. Expression of the HPPD-4 protein confers tolerance to HPPD inhibitor herbicides such as isoxaflutole.
The petitioner provided information to support the safety and long history of use of the donor organisms (B. thuringiensis and P. fluorescens) and the recipient organism (G. max 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 GMB151 was characterized by using a combination of whole genome sequencing (WGS), Junction Sequence Analysis (JSA), and directed sequencing (locus-specific PCR, DNA sequencing and analyses). The results of the analyses demonstrated the presence of a single transgenic locus which consists of a single copy of the T-DNA insert, lacking the 5' part of the P2x35S promoter of the hppdPf-4Pa gene.
Bioinformatics analyses were performed to assess potential toxicity and allergenicity of any putative peptides encoded by the translation of the six reading frames within the T-DNA insert and the flanking gDNA sequences. Open reading frames (ORFs) were defined by searching the encompassed nucleotide sequence for any initiation and stop codons producing a peptide of eight amino acids or greater in length. A total of 601 putative ORFs were identified. Subsequently, the translated amino acid sequences from the identified ORFs with a minimum size of 30 amino acids were used as query sequences in homology searches to known toxins and allergens. After elimination of duplicate ORFs, 115 unique sequences were selected for analysis.
Each query sequence was compared with all protein sequences present in the NCBI non-redundant database. The biological relevance of each match was assessed. No significant matches were found with any toxic protein from the database.
Two in silico approaches (an 8-mer search with 100 % sequence identity to an allergenic protein and an overall identity search with greater or equal to 35 % sequence identity calculated over at least 80 amino acids) were used to evaluate the potential amino acid sequence identity with known allergens contained in the public allergen database COMPARE (www.comparedatabase.org). The overall search showed no biologically relevant identities between the query sequences and any known allergenic proteins. One ORF matched with a contiguous 8-amino acid sequence present in the Cas s 5 allergen. No match with these proteins was found in the overall search and an 8-mer match in isolation is unlikely to indicate any potential cross-reactivity. Furthermore, translation of this 8-mer sequence is not possible due to the absence of a translation start codon and an RNA splice site
Stability of the T-DNA insert in the GMB151 genome was demonstrated by assessing individual GMB151 plants from five generations (T2, T4, T5, T6, BC2F3) by means of WGS and JSA. The results indicate that the T-DNA insert is intact and stable over all five generations of GMB151.
Segregation of the T-DNA insert was assessed in two F2 generations, two BC2F2 generations, and one BC1F2 generation of GMB151. Segregation ratios confirmed that the cry14Ab-1.b and hppdPf-4Pa genes contained within the T-DNA insert are inherited in a manner as expected for a single insertion. The results are consistent with Mendelian principles of inheritance and support the conclusion that the GMB151 genome contains a single T-DNA insert (albeit lacking the 5' part of the P2x35S promoter) integrated into a single chromosomal locus within the soybean genome.
4. Product Information
GMB151 differs from its traditional counterparts by the addition of two gene expression constructs: one for the cry14Ab-1.b gene which expresses a Cry14Ab-1 protein which results in resistance to the soybean cyst nematode (SCN), Heterodera glycines ichinohe, and the second for the hppdPf-4Pa gene which expresses a HPPD-4 protein conferring tolerance to HPPD inhibitor herbicides.
Protein expression levels of the Cry14Ab-1and HPPD-4 proteins in tissues of GMB151 were determined by Enzyme-Linked Immunosorbent Assay (ELISA) in field-grown soybean matrices from GMB151 plants treated and not treated with trait-specific herbicide grown at three field trials in the USA in 2016.
Mean (±SD) Cry14Ab-1 protein expression levels in not treated and treated seed at BBCH 89-99 (maturity) of GMB151 were 95.91 ± 43.11 µ/g DW and 83.14 ± 37.69 µ DW, respectively.
Mean (±SD) HPPD-4 expression levels in not treated and treated seed at BBCH 89-99 (maturity) of GMB151 were 4.46 ± 2.90 µ/g DW and 4.45 ± 3.57 µ/g DW, respectively.
Expression levels of the Cry14Ab-1 and HPPD-4 proteins in GMB151 were too low to allow for purification of sufficient quantities for use in safety assessment studies. Therefore, recombinant Cry14Ab-1 and HPPD-4 proteins were produced in B. thuringiensis and Escherichia coli, respectively. Equivalence of the plant- and microbially-produced proteins was established using a panel of analytical tests and assays including analysis of a Coomassie-strained SDS-PAGE; western blot analysis; glycostaining analysis; Mass Spectroscopy; N-terminal sequence analysis; and functional activity assays. Based on this panel of tests and analyses, the plant-produced Cry14Ab-1 and HPPD-4 proteins are considered equivalent to their microbially-produced counterparts. The microbially-produced proteins are therefore appropriate surrogates to be used for further assessment studies.
5. Dietary Exposure
It is expected that GMB151 will be used in applications similar to conventional soybean varieties. The petitioner does not anticipate a significant change in the food use of soybean with the introduction of GMB151.
6. Nutrition
Compositional data for GMB151 treated with hydroxyphenylpyruvate inhibitor herbicide, and its closely related conventional counterpart were obtained from eight field trials conducted across eight locations in the United States during the 2017 growing season. In each trial, four replicates of each entry were planted in a randomized complete block design.
Seed samples were harvested and analyzed for proximates and fibres, fatty acids, amino acids, minerals, vitamins, phytoestrogens, and anti-nutrients. These compositional components are in line with recommendations listed in the Organisation for Economic Co-operation and Development (OECD) consensus document on compositional considerations for new varieties of soybean. The analyses for each component were conducted using internationally approved and validated analytical methods.
The petitioner summarized the data as means and standard deviations, and conducted an inferential statistical analysis. If a statistically significant difference (P-value <0.05) between the modified and control seed was indicated, the nutritional relevance of the difference was determined through comparison to the expected range for conventional soybean as defined by values reported in the International Life Sciences Institute (now the Agriculture and Food Systems Institute) Crop Composition Database, the OECD consensus document, and the petitioner's submission based on compositional analysis of seed from commercially available reference varieties.
Statistically significant differences in GMB151 compared to the control were observed for the following components (GMB151 vs. control units): carbohydrates (34.5 vs. 35.2, % Dry Weight (DW)), neutral detergent fibre (15.7 vs. 16.3, % DW), palmitic acid (10.7 vs. 11.0, % Fatty Acid (FA)), oleic acid (23.4 vs. 21.8, % FA), linoleic acid (52.6 vs. 53.7, % FA), eicosenoic acid (0.191 vs. 0.182, % FA), behenic acid (0.382 vs. 0.331, % FA), lignoceric acid (0.098 vs. 0.111, % FA), calcium (2343 vs. 2547, mg/kg DW), copper (13.3 vs. 14.8, mg/kg DW), zinc (54.2 vs. 58.4, mg/kg DW), thiamin (1.46 vs. 1.80, mg/kg DW), pantothenic acid (9.50 vs. 8.64, mg/kg DW), folic acid (6.77 vs. 5.50, mg/kg DW), total daidzein (709 vs. 850, mg/kg DW), total genistein (703 vs. 808, mg/kg DW), total glycitein (134 vs. 160, mg/kg DW), total isoflavones (1546 vs. 1818, mg/kg DW), and phytic acid (1.60 vs. 1.45, % DW). All these observed differences in the composition of GMB151 were within their expected ranges for conventional soybean, respectively. As such, these differences were not considered to be a nutritional safety concern
Based on the information provided on the composition of GMB151 and control, there are no safety concerns with the use of GMB151 as a food in Canada from a nutritional perspective.
7. Toxicology
Safety of the novel proteins, Cry14Ab-1 and HPPD-4, is supported by the results of the submitted toxicity studies. These studies followed established OECD guidelines for the testing of chemicals. Safety data included bioinformatics searches, acute oral toxicity, heat stability, digestibility, and dietary exposure.
The genes for the novel proteins were derived from the non-pathogenic bacteria B. thuringiensis and P. fluorescens. Bioinformatics analyses of the amino acid sequences for the Cry14Ab-1 and HPPD-4 proteins were conducted to compare overall identity with known toxins, using the NCBI non-redundant database (version 2018_0506, 2018-04-28) and the in-house Bayer toxin database (BCS 2018 toxin database, version 18.1, 2018-02-15). Searches were performed June 07, 2018. No biologically relevant matches were identified for known toxins. Searches conducted on the complete T-DNA insert, based on putative proteins from ORFs, were also negative for known toxins.
Toxicity testing was conducted with surrogate microbially-produced proteins for Cry14Ab-1 and HPPD-4. These proteins are equivalent to the plant-derived proteins, based upon molecular weight, amino acid sequence, immunoreactivity, and functional assays.
Acute oral (gavage) toxicity studies in mice determined the No Observed Adverse Effect Level (NOAEL) is greater than 2000 mg/kg body weight (bw) for both Cry14Ab-1 and HPPD-4.
The Cry14Ab-1 and HPPD-4 proteins are sensitive to heat degradation, at temperatures that would be encountered during processing and cooking (≤ 75 °C). They are also likely to be easily digested after ingestion, as suggested by the results of in vitro simulated gastric fluid and intestinal fluid digestion assays. As a result, exposure to the novel proteins is expected to be negligible.
Estimated dietary exposures to the Cry14Ab-1 and HPPD-4 proteins were calculated by the petitioner (with the assumption that no loss or degradation occurs). The highest consumption occurred with acute exposure in children; 773.41 µ/kg bw for Cry14Ab-1 and 41.22 µ/kg bw for HPPD-4.
The margin of exposures (MOEs) for the novel proteins was calculated. The NOAEL of 2000 mg/kg bw observed in the acute oral toxicity studies in mice was compared to the acute dietary exposure in children of 773.41 µ/kg bw for Cry14Ab-1 and 41.22 µ/kg bw for HPPD-4. These calculations generated MOEs of 2,587 for Cry14Ab-1 and 48,780 for HPPD-4. These MOEs are considered sufficient from a safety perspective.
Based on the information provided, evaluators did not identify any safety concerns regarding the food use of GMB151 from a toxicological perspective.
8. Allergenicity
Both novel proteins, Cry14Ab-1 and HPPD-4, are sensitive to heat degradation, at temperatures that would be encountered during processing and cooking (≤ 75 °C). They also quickly degrade within 30 seconds in an in vitro simulated gastric fluid assay. These results suggest that exposure to the novel proteins is expected to be negligible.
Bioinformatics analyses of the amino acid sequences for the Cry14Ab-1 and HPPD-4 proteins were conducted to search for matches with greater than 35 % identity over a window of 80 amino acids and a search for sequence identity of eight contiguous amino acids. Searches were performed using the Comprehensive Protein Allergen Resource (version COMPARE 18, 2018-02-13), on June 7, 2018. The results did not detect any sequence homology matches with known allergens. Searches conducted on the complete T-DNA insert, based on putative proteins from ORFs, were also negative for known allergens.
Soybean is a priority allergen in Canada. Soybean products containing soy proteins, contain soybean allergens. Compositional analysis, including protein content, demonstrates that GMB151 is equivalent to conventional soybean. The similarity between GMB151 and conventional soybean in phenotypic and growth characteristics additionally supports the absence of off-target effects from the genetic modification or the expression of the novel proteins. This suggests that the amount of endogenous allergens is expected to be equivalent in GMB151 products compared to conventional soybean products.
The novel proteins expressed in GMB151 are not expected to pose an additional allergenic concern, due to lack of homology with known allergens and sensitivity to degradation.
Based on the information provided, evaluators did not identify any safety concerns regarding the food use of GMB151 from an allergenic perspective.
Conclusion:
Health Canada's review of the information presented in support of the food use of GMB151 does not raise concerns related to food safety. Health Canada is of the opinion that food derived from this soybean variety is as safe and nutritious as food derived from current commercial soybean varieties.
Health Canada's opinion deals only with the food use of GMB151. Issues related to its environmental release and 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.
(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
hc.bmh-bdm.sc@canada.ca
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