Novel food information: Maize event EH913

On this page

• Background
• Introduction
• Development of the modified plant
• Characterization of the modified plant
• Product information
• Dietary exposure
• Nutrition
• Chemistry/Toxicology
• Conclusion

Background

Health Canada has notified Helix Sementes e Mudas that it has no objection to the food use of maize event EH913. 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.

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

Introduction

Maize event EH913 was genetically modified to introduce gene expression cassettes for the insecticidal Cry1Da protein and the selectable marker phosphinothricin-N-acetyltransferase (PAT) protein. The Cry1Da protein acts to kill insect pests and the PAT (bar) protein confers glufosinate tolerance. This maize line was developed to be insect resistant to the lepidopteran maize pest Spodoptera frugiperda (Fall armyworm) and to Diatraea saccharalis (Sugarcane borer).

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 maize event EH913 was developed; how the composition and nutritional quality of maize event EH913 compares to non-modified maize; and what the potential is for maize event EH913 to be toxic or cause allergic reactions. Helix has provided data to demonstrate that their insect resistant maize is as safe as conventional maize varieties used as food in Canada.

The Food Directorate has a legislated responsibility for the pre-market assessment of novel foods and novel food ingredients, as detailed in Division 28 of Part B of the Food and Drug Regulations. Foods derived from Helix maize event EH913 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

The petitioner has provided information describing the methods used to develop maize event EH913 and data that characterize the inserted genetic material which provides insect resistance to Fall armyworm and Sugarcane borer as well as glufosinate tolerance. Maize event EH913 protects against pests due to the insertion of the known insecticidal tryptic core of the Cry1 family of proteins. These act on the larval stages of pests to exert toxicity through formation of pores in the larval gut which leads to lysis of cells and larval mortality. The PAT (bar) protein allows for selection of successful transformants via glufosinate tolerance.

Maize event EH913 was developed using Agrobacterium-mediated transformation of the Hi-II maize inbred line with the plasmid vector pEH001. This plasmid carries two expression cassettes. The first cassette contains the Cry1Da gene from Bacillus thuringiensis 1132C strain isolated in Brazil and which produces the 625 amino acid N terminal segment of the native truncated Cry1Da insecticidal protein from B. thuringiensis that is codon optimized for enhanced expression in maize. The second cassette contains the bar gene derived from Streptomyces hygroscopicus to produce phosphinothricin (PAT) protein that confers tolerance to glufosinate herbicide. Both donor species for the proteins are ubiquitous in soil and well described in the public domain.

The three primary genetic elements of the first Cry1Da cassette are a UBI promoter and associated intron from Zea mays that is used to initiate transcription, the truncated Cry1Da protein sequence for insect resistance, and the NOS transcription terminal polyadenylation site from Agrobacterium spp. as a termination signal. The primary elements of the second PAT cassette are the CaMV 35S promoter (double) from Cauliflower Mosaic Virus that begins transcription, the PAT protein coding sequence, and the TVSP transcription terminal polyadenylation site from Glycine max vegetative storage protein used to end transcription.

Characterization of the modified plant

Whole genome sequence analysis (both Illumina and PacBio), PCR, and bioinformatics were used to assess maize event EH913 to demonstrate insert and copy number, integrity of the inserted expression cassettes, absence of backbone sequences, and lack of unintended effects.

The results indicated that maize event EH913 contains one copy of the T-DNA at a single integration locus and with no detectable vector backbone sequence in the genome of maize event EH913. There was one additional "C" residue in the UBI intron sequence of cassette one, however this residue had no impact on any coding sequences and therefore no molecular relevance.

The inheritance of the genetic change across multiple generations was demonstrated using chi-square analysis to observe segregation ratios according to Mendelian inheritance principles. The genetic inheritance of the glufosinate tolerant phenotype was tested using three successive backcrosses with the L3 inbred. The resulting BC1, BC2, and BC3 generations segregated with the expected incidence of glufosinate tolerance. Both the glufosinate tolerance and insect resistance are found on the same T-DNA and thus inherited together.

The stability of the T-DNA in maize event EH913 was assessed by Illumina sequencing of the T-DNA and across the 5' and 3' junctions of the insert. The sequencing was conducted on DNA from four generations of EH913 spanning seven points on the EH913 breeding tree. The CLUSTAL alignment of the T-DNA sequences from the samples showed the expected sequence with no changes across the generations. This indicates that the T-DNA was maintained stably in the maize genome.

A search of the entire maize genome was conducted to look for potential new open reading frames. Bioinformatics assessments identified 6 potential open reading frames spanning the 5' and 3' junctions of the insertion. The results showed strong homology of both 5' and 3' flanking sequences to the maize genome and also showed no significant homology to transposable elements as well as no integration into a known maize transcriptional unit. The transcription of an unintended protein caused by the insertion is highly unlikely.

Product information

Maize event EH913 expresses two additional proteins, Cry1Da and PAT (bar). The Cry1Da protein is a 70 kDa truncated version and the PAT (bar) protein is 21 kDa. The characterization of the T-DNA showed that the genes for both proteins were intact and the expected protein sequences were inserted into the maize genome.

The donor organisms of the coding sequences of the two proteins, Bacillus thuringiensis (Cry1Da) and Streptomyces hygroscopicus (PAT(bar)) are ubiquitous in the environment and not commonly known for pathogenicity or allergenicity in humans or mammals. The Cry1 proteins, including Cry1Da, and PAT, both PAT(bar) from Streptomyces hygroscopicus and PAT(pat) from Streptomyces viridochromogenes, have a history of safe use in biotechnology crops.

The amino acid sequence of the 70 kDa Cry1Da protein in EH913 is 100% identical to the first 625 amino acids of the full length Cry1Da protein (133 kDa) found in the donor organism Bacillus thuringiensis ssp. Aizawai, and 99.4% identical to the corresponding sequence of the Cry1Da_7 protein available in the public domain and for which there are safety studies.

Glycosylation analysis of the Cry1Da protein and a microbial-produced protein showed no glycosylation for both proteins. Western blot characteristics using antibodies also showed no difference between plant produced and microbial produced Cry1Da. This supports the equivalency of the plant produced to microbial produced protein.

The PAT (bar) protein expressed in maize event EH913 has 100% amino acid identity to the PAT (bar) protein found in the donor organism and 85% amino acid homology to the PAT (pat) protein that has also been widely tested, approved, and grown in numerous countries.

Dietary exposure

The use of foods derived from Helix maize event EH913 is similar to other maize varieties already consumed in Canada and will be used in a variety of food applications. It is expected that there would be no change in the food use of maize derived foods caused by the presence of Helix maize event EH913.

Nutrition

Compositional data for maize event EH913, a conventional control non-GM hybrid from L85xL3 inbred lines, and four non-GM maize hybrid conventional varieties were obtained from grain sample harvested from seven field trials conducted in Brazil during the 2020-2021 growing season. In each trial, four replicates of each entry were planted in a randomized complete block design.

Grain samples were analysed using acceptable methods for proximates and dietary fibre, amino acids, fatty acids, minerals, vitamins and anti-nutrients as suggested by the Organisation for Economic Co-operation and Development Consensus Document on Compositional Considerations for New Varieties of Maize (Zea Mays): Key Food and Feed Nutrients, Anti-nutrients and Secondary Plant Metabolites (2002).

The Petitioner conducted statistical analyses comparing the composition of the modified maize with its conventional control. Where a statistically significant difference (P-value < 0.05) was identified, the nutritional relevance of the difference was determined through comparison to the concurrently grown commercial reference varieties.

Statistically significant differences between maize event EH913 and the conventional control were reported for the following analytes: protein, dietary fibre, methionine, palmitic acid, linoleic acid, calcium, zinc, beta-carotene, niacin, riboflavin and vitamin E (alpha-tocopherol). In all cases, the mean composition of maize event EH913 was within the range of values reported for the reference varieties and/or within the range of values reported in the Agriculture and Food Systems Institute Crop Composition Database (2020).

Based on the available information, maize event EH913 is similar in composition to its conventional counterpart.

Chemistry/Toxicology

Maize has a long history of safe food use. Two novel proteins (truncated Cry1Da and PAT) are produced by this genetically modified maize (EH913).

The toxicological safety rationale for the truncated Cry1Da novel protein included: the lack of mammalian toxicity of the source organism, Bacillus thuringiensis (Bt); the established non-mammalian mode of action of Cry proteins in general, and; a published report of the lack of acute oral toxicity in rodents of a modified (4 amino acid changes) full length Cry1Da (i.e. Cry1Da_7).

The source organism, Bacillus thuringiensis (Bt) occurs ubiquitously in the environment and is not commonly known for pathogenicity in mammals, including humans. For example, a Bt strain is registered as an insecticide in Canada, for use on food crops ^{Footnote 1}, based on an assessment that it lacked toxicity in oral rat studies, and did not produce toxic (to mammals) metabolites.^{Footnote 2}

Bt produces large parasporal crystals during sporulation. These crystals consist of various δ-endotoxins, mostly Cry proteins. The Cry proteins are cleaved in the insect gut by host proteinases, which give rise to 65- to 70-kDa activated toxins that form pores in the columnar cell brush border membranes, leading to the disruption of ion and metabolite transport and subsequent insect death. ^{Footnote 3} The toxic mode of action of Cry proteins towards certain insects would not be expected to occur in humans due to differences between species in the digestive tract (e.g. receptors not present in humans and more acidic environment of mammalian gut leads to degradation of Cry proteins). ^{Footnote 4} This is supported by the results of the simulated gastric fluid (SGF) assay for the truncated Cry1Da protein which showed quick (5 minutes) degradation of the protein.

After considering the similarities (e.g. similar insecticidal activity, 99.4% similarity in amino acid sequence, similar results in heat stability, SGF and simulated intestinal fluid (SIF) assays and observation of a similar stable break-down peptide in SIF consistent with the known mode of action of Cry proteins) between the truncated Cry1Da and Cry1Da_7 and a confirmation of protein equivalency, the no observed adverse effect level (NOAEL) of 5000 mg Cry1Da_7/kg bw (highest dose tested) was applied to the safety of the truncated Cry1Da protein in EH913 maize. Comparison of this NOAEL to a conservative estimate of exposure (0.00052 mg/kg bw) to the truncated Cry1Da, results in a margin of 5 100 000. This margin is considered adequate for safety.

The toxicological safety rationale for the PAT novel protein included: the lack of toxicity of the source organism, Streptomyces hygroscopicus; previously approved food crops containing PAT, and; a published report of the lack of acute oral toxicity in rodents of PAT.

The source organism, Streptomyces hygroscopicus occurs ubiquitously in the environment and is not commonly known for pathogenicity in mammals, including humans.

Several novel maize containing PAT protein have been previously assessed and authorized for sale in Canada.

After considering the similarity between the tested and notified PAT proteins and a confirmation of protein equivalency, the NOAEL of 2500 mg PAT/kg bw (highest dose tested) from the published summary of an acute oral toxicity study of purified PAT in mice ^{Footnote 5} was applied to the safety of the PAT protein in EH913 maize. Comparison of this NOAEL to a conservative estimate of exposure (0.0000043 mg/kg bw) to PAT from EH913 maize, results in a margin of 580 000 000. This margin is considered adequate for safety.

Based on the available data, from a toxicological perspective, maize event EH913 is considered as safe as conventional corn currently available on the Canadian market.

The sources of the genes (Bt and S.hygroscopicus) resulting in the two novel proteinsand the host organism (maize) have not previously been associated with food allergy. Dietary exposures to these novel proteins from EH913 maize are expected to be low (<1 µg/kg bw). No amino acid homology matches ^{Footnote 6} based on international criteria ^{Footnote 7} were observed between these novel proteins and known allergens. These novel proteins are likely to be inactivated during some corn cooking/processing conditions and quickly degraded in the stomach, based on heat stability and SGF assay results, resulting in little opportunity for interaction with the immune system and resulting allergic reactions. These novel proteins do not share characteristics associated with some known food allergens, such as stability in heat and SGF assays.

Based on the available data, from an allergenicity perspective, EH913 Maize is considered as safe as conventional corn currently available on the Canadian market.

Conclusion

Health Canada's review of the information presented in support of the food use of maize event EH913 does not raise concerns related to food safety. Health Canada is of the opinion that food derived from maize event EH913 is as safe and nutritious as food from conventional corn currently available on the Canadian market.

Health Canada's opinion deals only with the food use of maize event EH913.

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 and Nutrition Directorate
Health Products and Food Branch
Health Canada, PL2204A1
251 Frederick Banting Driveway
Ottawa, Ontario K1A 0K9
bmh-bdm@hc-sc.gc.ca