Novel food information: Short stature maize MON 94804

On this page

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

Background

Health Canada has notified Bayer CropScience Inc. (Bayer) that it has no objection to the food use of short stature maize – MON 94804 (MON 94804). The Department conducted a comprehensive assessment of this maize 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 the evaluation by Health Canada. This document contains no confidential business information.

Introduction

Bayer has developed a novel maize (Zea mays L.) variety, MON 94804 that which exhibits reduced overall plant height (i.e., a short stature) compared to conventional maize.

MON 94804 was developed through the introduction of a suppression cassette to down regulate the expression of gibberellic acid 20 oxidase proteins involved in the determination of plant height. The cassette contains a GA20ox_SUP sequence composed of an inverted repeat derived from the coding sequence of the endogenous maize genes, GA20ox3 and GA20ox5, which encode the gibberellic acid 20 oxidase 3 and 5 proteins^{Footnote 1}, flanked and separated by three Osa-miR1425 fragments derived from Oryza sativa (rice)^{Footnote 2}.

A cp4 epsps gene derived from Agrobacterium sp. strain CP4, was initially introduced into the transformed plant genome as a selection marker to identify successful transformants but was subsequently removed from the genome using Cre/lox technology. The history of safe use of Agrobacterium sp. strain CP4 (and the cp4 epsps gene) has been previously reviewed as part of the assessment of several glyphosate-tolerant plant products, including but not limited to glyphosate tolerant soybean 40-3-2, glyphosate tolerant soybean MON 89788, glyphosate tolerant sugar beet event H7-1, herbicide-tolerant sugar beet - KWS20-1, glyphosate tolerant alfalfa events J101 and J163, TruFlex Roundup Ready canola MON 88302, and herbicide tolerant HT4 maize - MON 87429.

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 MON 94804 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 has 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 MON 94804 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

MON 94804 was developed through Agrobacterium-mediated transformation of maize breeding line HCL301 with plasmid vector PV-ZMAP527892, based on the method described by Ye et al. (2022)^{Footnote 3}. Briefly, mature embryos were excised from a post-pollinated maize ear of HCL301. After co-culturing the excised mature embryos with Agrobacterium tumefaciens carrying the transformation plasmid vector PV-ZMAP527892, the mature embryos were placed on selection medium containing glyphosate to inhibit the growth of untransformed plant cells. Once transformed callus developed, the callus was placed on media conducive to shoot and root development. The rooted plants (R0) with normal phenotypic characteristics were selected and transferred to soil for growth and further assessment.

Leaf samples were collected from R0 plants. Genomic DNA was extracted from the leaf samples and used as templates for screening for the presence of transfer DNA (T-DNA) and absence of plasmid vector backbone sequences by real-time polymerase chain reaction (PCR) assays. The R0 plants carrying one copy of T-DNA were self-pollinated to produce R1 seed. Subsequently, the R1 population was screened for the presence of T-DNA and absence of plasmid vector backbone sequences by real-time PCR assays. Only plants that were homozygous positive for T-DNA and negative for plasmid vector backbone were selected for further development and their progenies were subjected to further molecular 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 the transformation plasmid vector PV-ZMAP527892. The T-DNA that was initially transformed into maize also contained a cp4 epsps selectable marker cassette flanked by loxP sites. R2 events were crossed with a line expressing the Cre recombinase protein (the Cre line was transformed with the vector PV-ZMOO513642) and screened for the absence of the cp4 epsps and cre genes using real-time PCR assays.

Cre/lox technology enables the removal of DNA sequence positioned between two excision targeting sequences called lox sites. In this instance, the Cre recombinase enzyme facilitates the excision of the selectable marker cassette which was inserted during the transformation as part of the T-DNA insertion that also included the GA20ox_SUP suppression cassette.

Subsequently, segregation, selection, and screening were used to isolate those plants that contained the GA20ox_SUP suppression cassette and lacked plasmid vector backbone and cp4 epsps selectable marker sequences from PV-ZMAP527892 and any sequences from the cre gene-containing plasmid vector PV-ZMOO513642.

After careful selection and evaluation of these events in the laboratory, greenhouse, and field, MON 94804 was selected as the commercial event based on superior characteristic efficacy, agronomic phenotypic, and molecular characteristics according to the general process described in Prado et al. (2014)^{Footnote 4}. Studies on MON 94804 were conducted to further characterise the genetic insertion and the expressed product, and to establish the food, feed, and environmental safety of MON 94804 relative to conventional maize.

Characterization of the modified plant

The molecular characterization of MON 94804 was conducted through bioinformatic mapping and subsequent analysis of short reads generated using Next-Generation Sequencing (NGS) technologies^{Footnote 5}. Additionally, directed sequencing (locus-specific PCR and DNA sequence analyses) was used to obtain the sequence of the T-DNA insert and the adjacent flanking DNA.

The results of these analyses confirmed the presence of the inserted T-DNA in at a single locus containing a single copy of the T-DNA, and the absence of any plasmid vector backbone sequence in the MON 94804 genome. Furthermore, the results of directed sequencing confirmed that the sequence of the inserted T-DNA is 100 % identical to the T-DNA sequence in the plasmid vector PV-ZMAP527892 except for the absence of the cp4 epsps selectable marker sequences as intended.

Alignments between the MON 94804 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 MON 94804. These results show that in MON 94804, the region at the T-DNA insertion site has a small deletion of 41 base pairs 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^{Footnote 6}.

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_2022 database, and a FASTA search against the TOX_2022 and PRT_2022 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, 2009^{Footnote 7}). 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_2022 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.

The results of these analyses indicate that no biologically relevant sequence similarities were observed between the translated putative polypeptides of the insert-to-flank junction sequences and allergen, toxins, or biologically active proteins associated with adverse effects for human or animal health. Other than the expression of the GA20ox_SUP micro RNA (miRNA), no evidence exists to indicate that any other sequence from the MON 94804 T-DNA insert is expressed or translated. The results of these bioinformatic analyses indicate that in the unlikely occurrence that any of the putative polypeptides were translated, none would share significant similarity or identity to known allergens, toxins, or other biologically active proteins that could affect human or animal health.

The genetic stability of the T-DNA insert in MON 94804 was demonstrated through multiple generations, mapping relative to the transformation plasmid (PV-ZMAP527892) and junction sequence identification were performed using NGS reads from DNA obtained from five breeding generations (F4, F4F1, F5, F5F1, F6) of MON 94804. The results of this analysis show that two identical junction sequences were detected for each MON 94804 generation, confirming the insertion of a single copy of the T-DNA insert at a single locus in the MON 94804 genome, and the consistency of these junctions in the mapping data across all generations tested demonstrates that this single locus is stably maintained throughout the MON 94804 breeding process.

The pattern of inheritance of the T-DNA insert in MON 94804 was demonstrated through segregation analysis of three generations (F4F1, F4F2, F4F3). A Pearson's chi-square (χ²) analysis was used to compare the observed segregation ratios of the MON 94804 T-DNA insert to the expected ratios. The results of the χ² analysis of the segregating progeny of MON 94804 indicated no statistically significant difference between the observed and expected segregation ratios of the MON 94804 T-DNA insert. These results support the conclusion that the MON 94804 T-DNA insert resides at a single locus within the maize genome and is inherited according to Mendelian principles of inheritance. These results are consistent with the molecular characterization data indicating that MON 94804 contains a single, intact copy of the T-DNA inserted at a single locus in the maize genome.

Based on the available data provided, the Bureau of Microbial Hazards (BMH) has no safety concerns regarding MON 94804 from a molecular perspective.

Product information

MON 94804 differs from its conventional counterpart by the expression of a GA20ox_SUP miRNA sequence composed of an inverted repeat derived from the coding sequence of the endogenous maize genes, GA20ox3 and GA20ox5, which encode the gibberellic acid 20 oxidase 3 and 5 proteins, flanked and separated by three Osa-miR1425 fragments derived from Oryza sativa (rice).

The expressed inverted repeat transcript is recognised by the endogenous RNA interference (RNAi) machinery of the plant, resulting in down-regulation of the targeted GA20ox gene expression. This suppression results in the reduction of bioactive gibberellic acid/gibberellin (GA) levels predominately in the stalk, leading to a reduction of internode length and consequently reduced overall plant height (i.e., a short stature) compared to conventional maize.

Northern blot analyses were performed to analyze the transcripts produced from the GA20ox_SUP suppression cassette in MON 94804 in multiple tissue types. Northern blot analyses showed that transcripts from the GA20ox_SUP suppression cassette are detected in over season leaf (OSL 1), over season root (OSR 1), stalk, and forage tissues in both populations of high and low molecular weight RNA molecules and in grain tissue in the population of low molecular weight RNA molecules, that are consistent with miRNA transcription and processing.

Northern blot analyses showed two populations (high and low molecular weight) of transcripts from the GA20ox_SUP suppression cassette, consistent with the expected pri-miRNA and mature miRNA, were present in MON 94804 OSL1, OSR1, stalk, and forage tissues. Only the low molecular weight RNA molecules, consistent with the mature miRNA, were detected in grain tissue. Northern blot analyses showed no detectable GA20ox_SUP transcripts in pollen tissue in both the high and low molecular weight RNA molecule populations.

Dietary exposure

It is expected that MON 94804 will be used in applications similar to conventional maize varieties. The petitioner does not anticipate a significant change in the food use of maize with the introduction of MON 94804.

Nutrition

Compositional data for MON 94804 and its conventional control were obtained from field trials conducted in the 2020 growing season across 5 locations in the United States. In each field trial, four replicates of each entry were planted in a randomized complete block design and grown under agronomic field conditions typical for the different growing regions.

Grain samples were harvested at physiological maturity and analyzed using acceptable methods, for proximates, fatty acids, amino acids, minerals, vitamins, anti-nutrients, and secondary metabolites. The data provided included all key nutrients, anti-nutrients, and secondary metabolites as suggested in the Organisation for Economic Development (OECD) "Consensus Document on Compositional Considerations for New Varieties of Maize (Zea mays): Key Food and Feed Nutrients, Anti-nutrients and Secondary Plant Metabolites" (2002)^{Footnote 8}.

The petitioner conducted a statistical analysis comparing MON 94804 with its conventional counterpart. Of the analytes measured, statistically significant differences were noted in the following analytes tested in the combined-site analysis: total dietary fibre (lower in MON 94804), and ferulic acid (lower in MON 94804). To determine if these statistically significant differences were nutritionally relevant, the MON 94804 analyte levels were further compared to expected ranges for conventional maize as defined by values reported in the Agriculture & Food Systems Institute Crop Composition Database and the OECD document referenced above. These differences were not considered to pose a nutritional safety concern as, in all cases, the composition of MON 94804 was within the expected ranges for conventional maize.

The Bureau of Nutritional Sciences (BNS) has not identified any nutritional concerns related to the proposed use of MON 94804.

Chemistry

Chemical contaminant residue data have not been provided as part of the novel food submission for MON 94804, nor are there maximum levels for contaminants specific to this food set out in Health Canada's List of Contaminants and Other Adulterating Substances in Foods and the List of Maximum Levels for Chemical Contaminants in Foods.

Given that the genetic modification for MON 94804 is morphological in nature, a literature search was conducted on reductions in plant height and potential implications for chemical contaminants and natural toxins. Overall, limited scientific information was available on this subject. Although not specific to maize, some information in the literature suggests that reduced plant height may correlate with the susceptibility of mycotoxins to certain grains (e.g., wheat). Given that reduced plant/internode height may impact moisture retention or air flow between the maize ears, fungal growth may become more favourable under certain environmental conditions. The petitioner was notified of these considerations and referred to the Codex Alimentarius Commission's Code of Practice to Reduce and Prevent Mycotoxin Contamination in Cereals (2016)^{Footnote 9}, which includes recommendations applicable to maize.

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, 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

No novel proteins are produced by this genetically modified food crop; the inserted genetic material produces an miRNA (i.e., the GA20ox_SUP miRNA). miRNAs are common components of the diet and have not been associated with toxicity.

Estimated human dietary exposures to the GA20ox_SUP miRNA are low. The highest estimate is 0.006 µg/kg body weight (bw) per day, assuming all corn eaten is MON 94804, and no degradation of miRNA during processing/cooking. Actual human dietary exposures are likely to be much lower than these estimates, because corn consumed is a mixture of varieties and miRNA is likely to be degraded during processing/cooking of maize.

Any miRNA consumed by humans which is not degraded during processing/cooking is unlikely to remain intact due to nucleases in saliva, and low pH levels and digestive enzymes in the stomach.

In the unlikely event that the GA20ox_SUP miRNA remains intact and is absorbed into human cells, there is no target for this miRNA within human cells, given that no nucleotide homology matches were observed between the GA20ox_SUP miRNA and human gene transcripts. The genes affected by this miRNA in maize are not present in humans or other animals.

MON 94804 is expected to be as safe as conventional maize in terms of food toxicity.

Based on the available information, the Pre-market Toxicology Assessment Section (PTAS) did not identify any toxicological food safety concerns with the use of MON 94804, as proposed.

Allergenicity

Almost all food allergens are proteins, and the GA20ox_SUP miRNA is not a protein. miRNAs have not been associated with allergenicity.

MON 94804 is expected to be as safe as conventional maize in terms of food allergenicity.

The PTAS did not identify any allergenic safety concerns with the proposed use of MON 94804.

Conclusion

Health Canada's review of the information presented in support of the use of MON 94804 does not raise concerns related to food safety.

Health Canada's opinion refers only to the food use of MON 94804. 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