Novel Food Information: Herbicide-tolerant sugar beet – KWS20-1

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Background

Health Canada has notified Bayer CropScience Inc. (Bayer) and KWS SAAT SE & Co. KGaA (KWS) that it has no objection to the food use of herbicide-tolerant sugar beet – KWS20-1 (KWS20-1). The Department conducted a comprehensive assessment of this sugar beet 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 characteristics.

The following provides a summary of the notification from Bayer and KWS and the evaluation by Health Canada. This document contains no confidential business information.

Introduction

Bayer and KWS have developed a novel sugar beet (Beta vulgaris) variety, KWS20-1 that which exhibits tolerance to dicamba (3,6-dichloro-2-methoxybenzoic acid), glufosinate (2-amino-4-(hydroxymethylphosphinyl) butanoic acid), and glyphosate (N-(phosphonomethyl)glycine)) herbicides.

KWS20-1 was developed through the introduction of three gene expression constructs for the expression of a dicamba mono-oxygenase (DMO) protein, a phosphinothricin N-acetyltransferase (PAT) protein, and a CP4 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) protein. Expression of the DMO, PAT, and CP4 EPSPS proteins confers tolerance to dicamba, glufosinate, and glyphosate herbicides, respectively.

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 KWS20-1 was developed, how the composition and nutritional safety of this variety compared to its unmodified comparator, and what the potential is for this variety to present a toxic or allergenic concern. Bayer and KWS have provided data to support that this variety is safe for use 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 Division 28 of Part B of the Food and Drug Regulations (Novel Foods). Foods derived from KWS20-1 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

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

Development of the Modified Plant

KWS20-1 was developed through Agrobacterium-mediated transformation of sugar beet breeding line 04E05B1DH05 with plasmid vector PV-BVHT527462, based on the method described by Lindsey and Gallois (1990)Footnote 1. Briefly, shoot segment tissues were excised from the embryos of germinated conventional seed. After co-culturing with Agrobacterium AGL1 strain carrying the transformation construct, the resultant meristematic tissues were placed on selection medium containing DL-phosphinothricin (PPT) to inhibit the growth of untransformed plant cells and timentin to inhibit the overgrowth of Agrobacterium. The resulting shoots were then placed in media conducive to root development. Rooted plantlets with normal phenotypic characteristics were selected. Events which passed the advancement criteria (e.g., the presence of a single, intact copy of the T-DNA insert, absence of vector backbone sequence, no insertion of repetitive regions or gene sequences) were selected and transferred to soil for growth and further assessment.

A single T0 plant generated through this transformation process was self-pollinated under an isolation bag to produce T1 seed. The plants from the T1 population were screened for the presence of plasmid vector PV-BVHT527462 T-DNA and the absence of vector backbone sequence by Kompetitive Allele Specific PCR (KASP) and Southern blot analyses. Twelve homozygous positive T1 plants were crossed by open pollination in an isolated field to generate T2 seed. Subsequently, T2 plants homozygous for plasmid vector PV-BVHT527462 T-DNA and negative for vector backbone sequences were selected for further development and their progenies were subjected to further molecular analysis, herbicide tolerance/efficacy, and phenotypic assessments.

As is typical of a commercial event production and selection process, hundreds of different transformation events (regenerants) were generated in the laboratory using PV-BVHT527462 and related vectors. After selection and evaluation of these events in the laboratory, greenhouse, and field, KWS20-1 was selected as the lead event based on superior agronomic, phenotypic, and molecular characteristics. Studies on KWS20-1 were initiated to further characterize the genetic insertion and the expressed novel products, and to establish its safety compared to commercially available sugar beet.

Characterization of the Modified Plant

The number of insertions, intactness of insertions, the presence/absence of unintentional insertions (e.g., vector backbone sequence), and the location of insertions in the KWS20-1 genome were determined using a combination of Southern blot analyses and directional sequencing.

The results of the Southern blot analyses confirmed the presence of the inserted T-DNA in a single locus containing a single copy of the T-DNA, and the absence of any plasmid vector backbone sequence in the KWS20-1 genome. results of directional sequencing confirmed that the sequence of the inserted T-DNA is 100 % identical to the T-DNA sequence in the plasmid vector PV-BVHT527462.

Alignments between the KWS20-1 consensus sequence (covering both the inserted T-DNA and its flanking 5' and 3' sequences) and the conventional control consensus sequence were performed to characterize the T-DNA insertion site in KWS20-1. These results show that in KWS20-1, the region at the T-DNA insertion site has a small deletion of 7-bp compared to the native sequence in the conventional control. Deletions like this are common events resulting from the double strand break repair mechanism, which constitutes a step in the T-DNA integration into the plant genome (Kleinboelting et al., 2015)Footnote 2.

Bioinformatics analyses were performed to assess the potential toxicity, allergenicity, or biological activity of the putative peptides encoded by the 5' and 3' inserted T-DNA/genomic DNA junctions. Sequences spanning the 5' and 3' junction sequences were translated from STOP-to-STOP codon in all six reading frames. Putative sequences were used as queries for both FASTA and eight (8)-amino acid (aa) sliding window searches against the AD_2021 database, and a FASTA search against the TOX_2021 and PRT_2021 databases.

The FASTA and 8-aa sliding window searches indicated that no biologically-relevant sequence similarities were observed between toxins, allergens, or other biologically-active proteins and the putative sequences.

As an additional conservative approach, sequences were identified as potentially cross-reactive if the linear identity is more than 35 % in a greater than 80-aa overlap for the junction sequences (Codex, 2009Footnote 3). For this search, the putative peptide query sequences were split into overlapping 80-aa query sequences. These 80-aa sequences were used in the FASTA36 search of the AD_2021 using default parameters with the following exception: an E-score cut-off of 100 was set as an initial high cut-off value to return a broad range of alignments. Resulting alignments were screened to see if any query yielded an alignment of 29-aa or more identities, the number required to surpass the threshold of 35 % identity thought to indicate potential for allergenic cross-reactivity (Codex, 2009). All alignments displaying at least 29-aa identities were captured and further assessed.

Other than the translation of the DMO, PAT, and CP4 EPSPS in KWS20-1, no evidence exists to indicate that any other sequence from the T-DNA is translated. In the unlikely occurrence that any of the putative peptides analyzed were translated, none would share significant similarity or identity with known toxins, allergens, or other biologically-active proteins that could affect human health.

The genetic stability of the T-DNA insert in KWS20-1 was demonstrated by performing Southern blot analysis on plants from three (3) breeding generations (T2, T3, and T4). Results of the analysis confirmed that the T-DNA insert is stably integrated over multiple generations of KWS20-1.

A one-generation segregation analysis was performed to study the inheritance pattern of the T-DNA insert in KWS20-1 sugar beet. The analysis was performed using the BC0S1 generation of KWS20-1 sugar beet obtained through the crossing of the T2 generation with an elite conventional sugar beet line followed by self-pollination of the hemizygous BC0 parent to produce the segregating BC0S1 population. A heterozygous KWS20-1 sugar beet hybrid and its conventional control were used as positive and negative controls, respectively.

Two dominant KASP assays were used to determine the presence or absence of the T-DNA in the segregating population. Two co-dominant KASP assays were used to determine the zygosity of plants in the BC0S1 generation, discriminating between homozygous positive, hemizygous positive, and homozygous negative (i.e., null segregants). Each of the two dominant and co-dominant KASP assays represent an independent analysis of the samples.

For the dominant KASP assays, results were consistent between the two assays. Statistical analysis of the results indicated that the observed output in the test reactions does not deviate from the expected 3:1 segregation ratio for the KWS20-1 sugar beet versus null segregant plants, respectively (p-value = 0.51). Therefore, these data show the segregation pattern of the KWS20-1 sugar beet T-DNA insert is consistent with the Mendelian inheritance of a single T-DNA insertion.

For the co-dominant KASP assays, results were consistent between the two assays. Statistical analysis of the results indicated that the observed output in the test reactions does not deviate from the expected 1:2:1 segregation ratio of homozygous positive, hemizygous positive, and homozygous negative plants, respectively (p-value = 0.79). These data show the segregation pattern of the KWS20-1 sugar beet T-DNA insert is consistent with the Mendelian inheritance of a single T-DNA insertion.

Based on the available data provided, the Bureau of Microbial Hazards (BMH) has no safety concerns regarding KWS20-1 from a molecular perspective.

Product Information

KWS20-1 differs from its conventional counterpart by the expression of a dicamba mono-oxygenase (DMO) protein, a phosphinothricin N-acetyltransferase (PAT) protein, and a CP4 5-enolpyruvulshikimate-3-phosphate synthase (EPSPS) protein.

The DMO protein, encoded by a dmo gene from Stenotrophomonas maltophilia strain DI-6, confers tolerance to dicamba herbicides by conversion of dicamba into non-herbicidal 3,6-dichlorosalicylic acid (DCSA) and formaldehyde. The DMO protein present in KWS20-1 is identical to the corresponding protein found in a number of approved events across several different crops that are currently commercialized and have a history of safe use including dicamba-tolerant soybean – MON 87708, dicamba and glufosinate-tolerant cotton – MON 88701, dicamba and glufosinate-tolerant maize – MON 87419, herbicide tolerant HT4 maize – MON 87429, dicamba-tolerant canola – MON 94100, and herbicide-tolerant soybean – MON 94313. The DMO protein has been fully characterized in the submitted safety studies for the pre-market safety assessments for MON 87708, MON 88701, and MON 87419. The identity of the DMO protein in KWS20-1 was confirmed by N-terminal peptide mapping.

The PAT protein, encoded by a pat gene from Streptomyces viridochromogenes, confers tolerance to the herbicidal active ingredient glufosinate-ammonium at current labelled rates by acetylating phosphinothricin to an inactive form. The PAT protein present in KWS20-1 is identical to the corresponding protein found in a number of approved events across several different crops that are currently commercialized and have a history of safe use including dicamba and glufosinate-tolerant maize – MON 87419, herbicide tolerant HT4 maize – MON 87429, glufosinate-tolerant maize – T25, insect-resistant and glufosinate-tolerant maize – TC 1507, insect-resistant and glufosinate tolerant maize – DAS-59122, herbicide-tolerant and pest-resistant maize – 4114, and glufosinate-tolerant soybean A5547-127. The identity of the PAT protein in KWS20-1 was confirmed by N-terminal peptide mapping.

The CP4 EPSPS protein is encoded by a cp4 epsps gene from Agrobacterium sp. strain CP4. Expression of the CP4 EPSPS protein confers tolerance to glyphosate herbicides through the ability of this protein to still function in the presence of the herbicide. This allows plants expressing the CP4 EPSPS protein to grow properly in the presence of the herbicide. The CP4 EPSPS protein present in KWS20-1 is identical to the corresponding protein found in a number of approved events across several different crops that are currently commercialized and have a history of safe use including, but not limited to, glyphosate-tolerant soybean – 40-3-2, glyphosate-tolerant soybean – MON 89788, glyphosate-tolerant sugar beet – H7-1, glyphosate-tolerant alfalfa – J101, glyphosate-tolerant alfalfa – J163, glyphosate-tolerant canola – MON 88302, and herbicide tolerant HT4 maize – MON 87429. The identity of the CP4 EPSPS protein in KWS20-1 was confirmed by N-terminal peptide mapping.

Expression of DMO, PAT, and CP4 EPSPS in two distinct tissues, roots and leaf/tops, of KWS20-1 were determined using validated enzyme linked immunosorbent assays (ELISAs). KWS20-1 was treated throughout the growing season with the appropriate herbicides considering their conferred herbicide tolerances and following accepted agronomic practices.

Tissues of KWS20-1 roots and leaf/tops were collected from four replicate plots planted in a randomized complete block design during the 2020 growing season from five field sites in the United States: 2 in Michigan, 2 in Idaho, and 1 in North Dakota. The field sites were representative of sugar beet-producing regions suitable for commercial production. Over-season leaf 1 (OSL), over-season leaf 2 (OSL2), tops, over-season root 1 (OSR1), over-season root 2 (OSR2), and over-season root 3 (OSR3; harvestable root) tissue samples were collected from each replicated plot in all five field sites.

The mean (±SE) DMO protein level in treated KWS20-1 sugar beet across all sites was highest in OSL1 at 140 (±6.5) µg/g dry weight (dw) and lowest in OSR3 at 12 (±0.53) µg/g dw.

The mean (±SE) PAT protein level in treated KWS20-1 sugar beet across all sites was highest in OSL1 at 25 (±1.2) µg/g dw and lowest in OSR3 at below the Limit of Quantification (<LOQ) (i.e., 0.125 µg/g dw for root).

The mean (±SE) CP4 EPSPS protein level in treated KWS20-1 sugar beet across all sites was highest in OSL1 at 590 (±33) µg/g dw and lowest in OSR3 at 100 (±5.9) µg/g dw.

Dietary exposure

It is expected that KWS20-1 will be used in applications similar to conventional sugar beet varieties. The petitioner does not anticipate a significant change in the food use of sugar beet with the introduction of KWS20-1. Seeing as there have long existed other herbicide-tolerant sugar beet events approved in Canada (e.g. glufosinate tolerant sugarbeet event T120-7 approved in 2000, glyphosate tolerant sugarbeet event H7-1 approved in 2005) and thus the existence of GM sugar beet would not be a new concept to consumers.

Nutrition

Sugar beet roots are processed into white sugar, pulp, and molasses for food, feed, or industrial applications and rarely used as a raw commodity. Sugar beets have a sucrose content of approximately 15-22 % depending on climate, soil type, variety, and cultivation methods. The main purpose of sugar beet processing is sugar (sucrose) recovery. Sugar beet tops are usually not consumed by humans; therefore, only the data pertaining to sugar beet roots were assessed.

Compositional data for genetically modified KWS20-1 and a genetically similar conventional control were obtained from root samples harvested from five field trials conducted in the US during the 2020 growing season. Each field trial included modified and conventional sugar beet in a randomized complete block design with four replicates. Samples (n=20) were analyzed using acceptable methods for proximates and fibres, amino acids, minerals, and secondary metabolites.

The data provided was for all 30 key components including moisture, protein, total fat, ash, amino acids (18), carbohydrates (by calculation), sucrose, crude fibre and pectin, phosphorus, potassium, sodium, and oleanolic acid (a secondary metabolite), as per the Organisation for Economic Co-operation and Development (OECD) "Consensus document on compositional considerations for new varieties of sugar beet: Key food and feed nutrients and antinutrients" (2002).

Statistically significant differences (P<0.05) between the modified sugar beet and the conventional control were observed for lysine, proline, serine, threonine, total fat, ash, phosphorus, and potassium. However, the composition of KWS20-1 was within the range observed for reference varieties and the Agriculture & Food Systems Institute (AFSI) Crop Composition Database reference range for each analyte. There were no differences for the other key components between KWS20-1 and the control non-modified sugar beet.

The Bureau of Nutritional Sciences (BNS) has not identified any nutritional concerns related to the proposed use of KWS20-1.

Chemistry

Chemical contaminant residue data have not been provided, nor have any unique contaminant considerations been identified with respect to KWS20-1. As well, there are no maximum levels for contaminants specific to sugar beet set out in Health Canada's List of Contaminants and Other Adulterating Substances in Foods or the List of Maximum Levels for Chemical Contaminants in Foods.

Sugar is a standardized food in Division 18 of the Food and Drug Regulations which specifies a high level of purity of not less than 99.8% sucrose. Sucrose must contain no more than 1 mg/kg arsenic and 0.1 mg/kg lead according to the Food Chemicals Codex (FCC). Additionally, sugar beets are highly processed as they are refined into sugar; processing steps are reported in the literature to reduce the concentrations of mycotoxins and trace elements that may be present in raw sugar.

As with any food or food ingredient sold in Canada, it is the responsibility of the food manufacturer to ensure that its use does not result in a violation of Section 4(1)(a) and (d) of the Food and Drugs Act, which states that no person shall sell an article of food that has in or on it any poisonous or harmful substance or is adulterated. If an elevated concentration of any chemical contaminant is found in any type of food, including sugar beet or sugar derived from it, the Bureau of Chemical Safety (BCS) may conduct a human health risk assessment to determine if there is a potential safety concern and whether risk management measures are required.

Toxicology

To support safety, the petitioner demonstrated that the novel proteins expressed (DMO, PAT, and CP4 EPSPS) are equivalent to similar novel proteins previously approved by Health Canada in various crops, which was confirmed by the BMH. The previous assessments determined that the novel proteins expressed are not acutely toxic, are degraded by heat treatment, and are readily digested. The petitioner provided updated bioinformatic analyses for the novel proteins and for putative proteins encoded by the insert junctions. The petitioner demonstrated the novel proteins are expressed at low levels in the mature roots of KWS20-1, which is the food source.

The DMO, PAT, and CP4 EPSPS proteins are not toxic, based on acute oral toxicity testing in mice. No adverse effects were observed with acute doses of 140 mg/kg body weight (bw) (MON 87708) or 283 mg/kg bw (MON 88701) for the DMO protein; 1086 mg/kg bw for the PAT protein (MON 88701); or 572 mg/kg bw for the CP4 EPSPS protein (H7-1 sugar beet).

The DMO, PAT, and CP4 EPSPS proteins are heat labile. The DMO protein is inactivated (as determined by loss of enzyme function in an assay and loss of protein integrity with SDS-PAGE analysis) when exposed to 55°C for 15 min (MON 88701; MON 87429). The PAT protein is inactivated when exposed to 75°C for 15 min and the CP4 EPSPS protein is inactivated when exposed to 75°C for 15 min (MON87429). These results indicate that inactivation of the proteins is expected to occur from temperatures encountered during food processing and cooking.

The DMO, PAT, and CP4 EPSPS proteins are readily degraded by digestive enzymes (as determined by SDS-PAGE analysis), when tested in vitro using simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) in conditions similar to those found in the human gastrointestinal tract. When exposed to SGF, digestion occurs within 30 seconds for these proteins (MON 87429).

The petitioner conducted bioinformatic analyses on the novel proteins and the putative peptides encoded by the insert junctions, comparing the amino acid sequences to those of known toxins. Similarity was assessed using a FASTA search against the toxin database TOX_2021 (a subset of sequences derived from the Swiss-Prot database https://www.uniprot.org/, searched January 5, 2021) and the GenBank protein database (PRT_2021). No relevant matches were found.

The expression levels of the novel proteins were determined by enzyme linked immunosorbent assays (ELISAs) in leaf and root tissues. The highest mean novel protein levels were found in the leaf. The lowest mean novel protein levels were found in the mature root. The mean values in the root were 12 µg/g dw for DMO, below the level of detection (0.125 µg/g dw) for PAT, and 100 µg/g dw for CP4 EPSPS.

The petitioner notes that all detectable levels of protein are removed during processing of the sugar beet roots into sugar and molasses. Sugar beet pulp maintains some of the proteins but is not used in human food products. Dietary exposure to the novel proteins is considered to be negligible.

Based on the available information, the Pre-market Toxicology Assessment Section (PTAS) did not identify any toxicological food safety concerns with the use of KWS20-1, as proposed.

Allergenicity

The petitioner conducted bioinformatic analyses on the novel proteins and the putative peptides encoded by the insert junctions, comparing the amino acid sequences to those of known allergens. Both FASTA and amino acid sliding window searches were conducted against the allergen database COMPARE (https://comparedatabase.org; searched February 1, 2021). No relevant FASTA matches were found. Additionally, no alignments met or exceeded the Codex thresholdFootnote 4 of greater than 35% over 80 amino acids, and no eight amino acid matches were identified. No similarity to known allergens was identified.

The novel proteins are sensitive to heat, showing degradation at temperatures associated with food processing and cooking (≥ 75°C), and are readily digested in simulated gastric fluid (within 30 sec). Additionally, the novel proteins are expressed at low levels in the mature roots of KWS20-1 and protein is removed during processing into sugar and molasses. Therefore, dietary exposure to the novel proteins is expected to be negligible.

The PTAS does not consider KWS20-1 to pose any additional allergenic health concerns in comparison to conventional sugar beet.

Conclusion

Health Canada's review of the information presented in support of the use of KWS20-1 does not raise concerns related to food safety.

Health Canada's opinion refers only to the food use of KWS20-1. Issues related to its use as animal feed have been addressed separately through existing regulatory processes in the Canadian Food Inspection Agency.

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.

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
bmh-bdm@hc-sc.gc.ca

Footnotes

Footnote 1

Lindsey, K. and Gallois, P. 1990. Transformation of Sugarbeet (Beta vulgaris) by Agrobacterium tumefaciens. Journal of Experimental Botany 41 (226): 529-536.

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Footnote 2

Kleinboelting, N., Huep, G., Appelhagen, I., Viehoever, P., Li, Y., and Weisshaar, B. 2015. The Structural Features of Thousands of T-DNA Insertion Sites Are Consistent with a Double-Strand Break Repair-Based Insertion Mechanism. Molecular Plant 8:1651–1664.

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Footnote 3

Codex Alimentarius. 2009. Foods derived from modern biotechnology. Codex Alimentarius Commission, Joint FAO/WHO Food Standards Programme, Food and Agriculture Organization of the United Nations, Rome, Italy.

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Footnote 4

https://apps.who.int/iris/bitstream/handle/10665/340572/WHO-FOS-2001.01-eng.pdf?sequence=1&isAllowed=y

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