Novel Food Information: Abiotic Stress and Herbicide Tolerant HB4 Soybean (IND-00410-5)

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Background:

Health Canada has notified Bioceres Crop Solutions Corp. that it has no objection to the food use of Abiotic Stress and Herbicide Tolerant HB4 Soybean (IND-00410-5) (i.e., HB4 soybean). This event was initially submitted to the Department as an asset owned by Verdeca LLC. During the assessment of this event, the ownership of this asset was transferred to Bioceres Crop Solutions Corp. 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 original notification from Verdeca LLC. and the evaluation by Health Canada and contains no confidential business information.

1. Introduction

Verdeca LLC. has developed a genetically modified Glycine max L. (soybean) variety which exhibits increased tolerance to environmental stresses (resulting in reduced crop yield loss) and tolerance to glufosinate-ammonium herbicides.

HB4 soybean was developed through the introduction of two genes: a HaHB4 gene encoding a transcription factor belonging to the sub-family I of HD-Zip family (HAHB4), and a bar gene encoding a phosphinothricin acetyltransferase (PAT) 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. Verdeca LLC. has provided data that demonstrate that HB4 soybean 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 HB4 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 HB4 soybean and molecular biology data that characterized the genetic change, which results in increased tolerance to environmental stresses (resulting in reduced crop yield loss) and tolerance to glufosinate-ammonium herbicides.

HB4 soybean was developed through Agrobacterium-mediated transformation of soybean variety Williams 82 with a transformation vector pIND2-HB4. The pIND2-HB4 vector contains a construct (T-DNA) of the HaHB4 gene, encoding a transcription factor belonging to the sub-family I of HD-Zip family (HAHB4), and the bar gene, encoding a phosphinothricin acetyltransferase (PAT) protein.

The coding sequence for the HAHB4 protein was isolated from sunflower (Helianthus annuus L.). The HAHB4 protein belongs to a large class of transcription factors, which are present in multiple plant species and are regulated by response to different environments. Increases in the mRNA level of HaHB4 have been observed following exposure to different stressful environmental stimuli.

The coding sequence for the PAT protein was isolated from the bacterium Streptomyces hygroscopicus. Expression of the PAT protein within the cell detoxifies (i.e., acetylates the L-isomer of phosphinothricin; L-PPT), and thereby makes the plant tolerant to glufosinate-ammonium herbicides. During the development of HB4 soybean, the bar gene was used as a selection marker for successful transformants. However, it was found that expression of the PAT protein additionally conferred tolerance to glufosinate-ammonium herbicides at levels used in cultivation.

The petitioner provided information to support the safety and long history of use of the donor organisms (H. annuus L. and S. hygroscopicus) 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 HB4 soybean 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 HB4 soybean genome. Results of the Southern blot analysis also confirmed the absence of all vector backbone sequence in the HB4 soybean genome.

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. Seventy-four (74) peptides of up to 177 amino acids in length were identified.

The toxigenic and allergenic potential of each of these peptides was assessed by sequence comparison with known toxins in the NCBI database and Animal Toxin Database (ATDB) and known allergens in the AllergenOnline database. None of the putative peptides showed any significant homology with known toxins or allergens.

Stability of the T-DNA insert in the HB4 soybean genome was demonstrated by assessing individual HB4 soybean plants from four generations (T1, T3, T5, and T6) by means of PCR. The results indicate that the T-DNA insert is intact and stable over all four generations of HB4 soybean.

Segregation ratios confirmed that the HaHB4 and bar 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 HB4 soybean genome contains a single T-DNA insert integrated into a single chromosomal locus within the soybean genome.

4. Product Information

HB4 soybean differs from its traditional counterparts by the addition of two gene expression constructs: one for the HaHB4 gene which expresses a HAHB4 protein conferring increased tolerance to environmental stresses (resulting in reduced crop yield loss), and the second for the bar gene which expresses a PAT protein conferring tolerance to glufosinate-ammonium herbicides.

Protein expression levels of HAHB4 protein in tissues of HB4 soybean were determined by the absolute quantification (AQUA) method of protein quantification by targeted liquid chromatography-tandem mass spectrometry (LC MS/MS). In spite of the very high sensitivity of this method, HAHB4 protein was only detected in two forage tissue samples from two United States (US) field trials (5 and 4 ng/g dry weight (DW), respectively). Low expression of the HAHB4 protein may be due to temporal, environmental, localized expression, and/or active regulatory control, as has been described for related HD Zip transcription factors.

Protein expression levels of PAT protein were measured in forage and seed tissues of HB4 soybean grown in the 2012-2013 growing season using enzyme-linked immunosorbent assay (ELISA). The highest value measured in HB4 soybean seed tissue samples was 69.05 µ/g fresh weight (FW) and 12.68 µ/g FW in forage samples.

Protein expression levels of PAT protein in leaf, stem, root, flower, young pod, and seed tissues collected during the 2017 US growing season were measured using ELISA. PAT protein levels were similar in all tissues, ranging from 24 to 43 µ/g FW.

Due to the extremely low expression of the HAHB4 protein in HB4 soybean, the plant-derived protein could not be purified in sufficient quantities for further testing. The petitioner designed a recombinant version of the HAHB4 protein for overexpression in an Escherichia coli expression system. The E. coli-produced HAHB4 protein was designed to have the identical primary amino acid sequence as the plant-produced HAHB4 protein. Equivalency between the plant-derived protein and the E. coli-produced protein was demonstrated using liquid chromatography mass spectrometry (LC M/S), MALDI-TOF mass detecting, N-terminal sequencing, and SDS-PAGE.

5. Dietary Exposure

It is expected that HB4 soybean 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 HB4 soybean.

6. Nutrition

Five field trials were used to collect samples for compositional analysis of HB4 soybean and its control (Williams 82) counterpart. The field trials were conducted in major soybean production areas of the USA in 2013. These growing regions overlap with Canadian soybean growing regions. HB4 soybean and control were grown at all sites. Each of the groups was replicated four times in a randomized complete block design. In addition, compositional analysis was conducted on several commercial reference varieties at each site. Typical commercial agriculture production practices were used for the field trials.

Soybean seed samples were analyzed for moisture, protein, fat, ash, carbohydrate, acid detergent fiber (ADF), neutral detergent fiber (NDF), crude fibre, calcium, phosphorous, fatty acids, vitamin E and K1, amino acids, and anti-nutrients (daidzein, genistein, glycitein, stachyose, raffinose, phytic acid, lectin, and trypsin inhibitors). Forge samples were also analyzed for moisture, protein, fat, ash, carbohydrate, and ADF and NDF.

A total of 44 analytes in seed and 7 in forage were analyzed. Statistical analysis was completed using SAS software and statistical differences were established at a 5 % level of significance. The analysis of HB4 soybean and control showed six statistically significant differences in the combined site analysis which occurred in: stearic acid, oleic acid, arachidic acid, vitamin K1, daidzein, and glycitein. In all cases, the differences were small, and the analyte levels were within the references ranges observed for commercial soybean varieties and/or as reported in the scientific literatureFootnote 1.

Based on the information provided on the composition of HB4 soybean and control, there are no safety concerns with the use of HB4 soybean as a food in Canada from a nutritional perspective.

7. Toxicology

The safety of the PAT protein is well documented in the public literature and further supported by the previous assessments of nine genetically modified crops expressing this protein. The equivalence of the PAT protein expressed in HB4 soybean and the PAT protein as it is described in the literature and previous assessments was confirmed. As such, the safety of the PAT protein produced from the bar gene is well-established and thus no new toxicological studies were required to support the safety of PAT expressed in HB4 soybean.

The main argument for the safety of the HAHB4 protein relates to a lack of exposure. The petitioner analysed the expression levels of the HAHB4 protein in soybean seeds. Two different methods of analyses were conducted and both failed to detect the protein in soybean seed using sensitive methods (LC-MS/MS, LOD = 0.026 µ/g dry weight (DW); TSQ MS, LOD = 0.007 µ/g DW). The very low potential for exposure suggests that the HAHB4 protein would not pose a toxicological concern to consumers.

The most conservative dietary exposure estimate was calculated by evaluators. The exposure assessment was based on the fact that infants are the highest consumers of soy on a body weight (bw) basis and on the assumption that the HAHB4 protein is expressed at the LOD. Using infant formula consumption values and assuming that soy protein is added at the maximum permitted level, it was estimated that infants aged 0-6 months would consume the HAHB4 protein up to 0.13 µ/kg bw per day. Therefore, even at the most conservative estimate, it was concluded that the potential exposure to the novel protein is considered to be extremely low and is not expected to pose a safety concern.

The petitioner provided analyses to determine whether the protein is digested in simulated gastric fluid (SGF) and degraded by heat. The results indicated that the HAHB4 protein is readily digested in SGF within 0.5 minutes, but is not significantly degraded by heat. Although bands of small proteins (~5 kDa) were still visible up to 30 minutes after exposure to SGF, they were not considered to be toxicologically significant since they showed no amino acid sequence homology to known protein toxins. The vast majority of the protein was instantly degraded suggesting that the intact protein would not be absorbed and would therefore not be expected to pose a toxicological concern.

The petitioner identified 74 putative open reading frames (ORFs) which could in theory result in the translation of a new protein. To preclude this possibility, the petitioner identified the putative peptides which could be expressed by these ORFs by searching the NCBI database and also compared their amino acid sequences to known protein toxins in the ATDB. All 74 putative peptides were hypothetical in nature (i.e., predicted by sequence analysis) and none matched known toxins in the databases. Based on this analysis, it is unlikely that any unintended peptides of concern would be expressed in HB4 soybean.

Based on the information provided, evaluators did not identify any safety concerns regarding the food use of HB4 soybean from a toxicological perspective.

8. Allergenicity

The safety of the PAT protein is well documented in the public literature and further supported by the previous assessments of nine genetically modified crops expressing this protein. The equivalence of the PAT protein expressed in HB4 soybean and the PAT protein as it is described in the literature and previous assessments was confirmed. As such, the safety of the PAT protein produced from the bar gene is well-established and thus no new allergenic studies were required to support the safety of PAT expressed in HB4 soybean.

Using multiple databases (AllergenOnline, Allergone.org, SDAP Database), the petitioner analysed the amino acid sequence as well as the protein's structural components to determine whether the HAHB4 protein resembled any known allergens. No relevant matches were found. Additionally, the HAHB4 protein is not glycosylated – a common feature of allergenic proteins (Ladics et al., 2018Footnote 2).

The petitioner demonstrated that the HAHB4 protein is rapidly digested (within 0.5 minutes) in a simulated gastric fluid (SGF) assay. This suggests that even if the protein were to be consumed, it would not reach immunological tissue in the gastrointestinal tract and would not be expected to elicit an allergic reaction. Faint bands representing small protein fragments (~5 kDa) remaining up to 30 minutes after digestion were not expected to pose a concern as there were no sequence homologies to known allergens.

Soybean is a priority allergen in Canada. The petitioner conducted an analysis of the levels of several known endogenous allergens expressed in HB4 soybean compared to its parental line as well as several other commercially available soybean lines. The results suggested that the genetic modifications introduced into HB4 soybean did not significantly alter the levels of endogenous allergens (i.e., the levels of endogenous allergens were within the documented ranges for soybean).

The amino acid sequence of the 74 putative peptides that could theoretically be expressed by putative ORFs identified in HB4 soybean were compared to sequences of known allergens in the AllergenOnline database. No significant matches were found. Therefore, in the unlikely event that these putative peptides would be expressed, they would not be expected to pose an allergenic concern.

Based on the information provided, evaluators did not identify any safety concerns regarding the food use of HB4 soybean from an allergenic perspective.

Conclusion:

Health Canada's review of the information presented in support of the food use of HB4 soybean 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 HB4 soybean. 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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