Novel food information: Herbicide Tolerant HT4 Maize – MON 87429

On this page

• Background
• 1. Introduction
• 2. Development of the Modified Plant
• 3. Characterization of the Modified Plant
• 4. Product Information
• 5. Dietary Exposure
• 6. Nutrition
• 7. Chemistry/Toxicology
• Conclusion

Background:

Health Canada has notified Bayer CropScience Inc. that it has no objection to the food use of herbicide tolerant HT4 maize – MON 87429. 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 traits.

The following provides a summary of the original notification from Bayer CropScience Inc. and the evaluation by Health Canada and contains no confidential business information.

1. Introduction

Bayer CropScience Inc. has developed a genetically modified Zea mays (maize) variety which exhibits exhibits tolerance to dicamba (3,6-dichloro-2-methoxybenzoic acid) herbicide; glufosinate (2-amino-4-(hydroxymethylphosphinyl) butanoic acid) herbicide; quizalofop (2-[4-(6-chloroquinoxalin-2-yl) oxyphenoxy] propanoic acid) herbicide; and 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide. MON 87429 also produces the 5-enolpyruvylshikimate-3-phosphate synthase protein from Agrobacterium sp. strain CP4 (CP4 EPSPS). MON 87429 utilizes an endogenous maize regulatory element to target CP4 EPSPS mRNA for degradation in tassel tissues, resulting in reduced CP4 EPSPS protein expression in pollen. Appropriately timed glyphosate applications to MON 87429 produce a non-viable pollen phenotype and allow for desirable cross pollination in female inbred parents.

MON 87429 was developed through the introduction of four genes: a dmo gene encoding a dicamba mono-oxygenase (DMO); a pat gene encoding a phosphinothricin acetyltransferase (PAT [pat]); a ft_t gene encoding a a-ketoglutarate dependent dioxygenase (FT_T); and a cp4 epsps gene encoding a 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS).

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 maize variety was developed; how the composition and nutritional quality of this variety compared to non-modified maize varieties; and the potential for this maize variety to be toxic or cause allergic reactions. Bayer CropScience Inc. has provided data that demonstrate that MON 87429 is as safe and of the same nutritional quality as traditional maize 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). MON 87429 is considered a novel food 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 MON 87429 and the molecular biology data that characterize the genetic change, which results in tolerance to dicamba, glufosinate, quizalofop, and 2,4-D herbicides, and tissue-specific glyphosate herbicide tolerance.

MON 87429 was developed through Agrobacterium-mediated transformation of maize variety LH244 with a transformation vector (PV-ZMHT519224) containing a construct (T-DNA) of a dmo gene encoding a dicamba mono-oxygenase (DMO), a pat gene encoding a phosphinothricin acetyltransferase (PAT [pat]), a ft_t gene encoding a a-ketoglutarate dependent dioxygenase (FT_T), and a cp4 epsps gene encoding a 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS).

The coding sequence for the DMO protein was isolated from the bacterium Stenotrophomonas maltophilia strain DI-6. The DMO protein catalyzes the NADH-dependent oxidative demethylation of dicamba, detoxifying the herbicide. Expression of the DMO protein thereby makes the plant tolerant to the dicamba herbicide. In MON 87429, the DMO protein is targeted to chloroplasts by a chloroplast transit peptide (CTP) to allow co-localization with the endogenous reductase and ferredoxin enzymes that supply electrons for the DMO methylation reaction.

The coding sequence for the PAT (pat) protein was isolated from the bacterium Streptomyces viridochromogenes. The PAT (pat) protein catalyzes the acetylation of the L-isomer of phosphinothricin (L-PPT), detoxifying the herbicide. Expression of the PAT (pat) protein thereby makes the plant tolerant to glufosinate herbicide.

The coding sequence for the FT_T protein is a modified version of the Rdpa gene from the bacterium Sphingobium herbicidovorans. The FT_T protein is an a-ketoglutarate dependent dioxygenase that catalyzes the degradation of phenoxypropionate herbicides. Expression of the FT_T protein thereby makes the plant tolerant to 2-amino-4-(hydroxymethylphosphinyl) butanoic acid) herbicide; quizalofop (2-[4-(6-chloroquinoxalin-2-yl) oxyphenoxy] propanoic acid) herbicide; and 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide.

The coding sequence for the CP4 EPSPS protein was isolated from Agrobacterium sp. CP4. The CP4 EPSPS protein catalyzes the sixth reaction in the shikimate biosynthetic pathway. This enzyme is inactivated by the glyphosate herbicide, however the CP4 EPSPS is resistant to glyphosate and continues to function in the herbicide’s presence. Expression of the CP4 EPSPS protein allows the plant to grow in the presence of glyphosate. As mentioned previously, MON 87429 uses an endogenous regulatory element that will target CP4 EPSPS mRNA only in tassel tissues, rendering non-viable pollen in MON 87429 plants treated with glyphosate, while the remainder of the plant expresses the CP4 EPSPS protein and thus can withstand the glyphosate treatment.

The petitioner provided information to support the safety and historical use of each donor organism (S. maltophilia, S. viridochromogenes, S. herbicidovorans, and Agrobacterium tumefaciens) and the recipient organism (Z. mays L.). None of these organisms pose a health safety concern. Furthermore, the DMO, PAT (pat), and CP4 EPSPS proteins have been previously assessed by Health Canada in maize, cotton, and soybean (for DMO); maize and soybean (for PAT [pat]); and maize, canola, sugarbeet, alfalfa, and cotton (for CP4 EPSPS).

3. Characterization of the Modified Plant

The number of integration sites of the T-DNA insert in MON 87429 was characterized using a combination of whole genome sequencing (WGS) and directed sequencing (locus-specific PCR, DNA sequencing and analyses). Based on these analyses, it was demonstrated that MON 87429 contains a single copy of the T-DNA inserted into a single locus. This conclusion is confirmed by the sequencing and analysis of overlapping PCR products from this locus.

Genetic stability of the T-DNA insert in the MON 87429 genome was demonstrated by assessing individual MON 87429 plants from five generations (R3, R3F1, R4, R4F1, and R5) by means of WGS analyses. The results indicate that the T-DNA insert is intact and stable over all five generations of MON 87429. Chi-square (χ2) analysis of the segregation data for three backcrossed generations was performed to test the hypothesis that the T-DNA insert in MON 87429 is inherited in a manner that is predictable according to Mendelian principles and is consistent with insertion into a single chromosomal locus within the maize genome. The results are consistent with Mendelian principles of inheritance and support the conclusion that the MON 87429 genome contains a single T-DNA insert integrated into a single chromosomal locus within the maize genome.

4. Product Information

MON 87429 differs from its traditional counterparts by the addition of four genes: a dmo gene encoding a dicamba mono-oxygenase (DMO); a pat gene encoding a phosphinothricin acetyltransferase (PAT [pat]); a ft_t gene encoding a a-ketoglutarate dependent dioxygenase (FT_T); and a cp4 epsps gene encoding a 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS).

Expression levels of the DMO, PAT (pat), FT_T, and CP4 EPSPS proteins in MON 87429 were analyzed using a validated multiplexed immunoassay method. Expression values were reported on a mg/g dry weight (DW) basis in maize tissues (i.e., forage, forage-root, grain, over season leaf ([OSL] 1 and OSL 4, and over season root [OSR] 1) collected from herbicide-treated MON 87429 produced in United States field trials during 2017.

The mean DMO protein level in MON 87429 across all sites was highest in OSL 1 at 35 mg/g DW and lowest in OSR 1 at 2.3 mg/g DW. The mean DMO protein level in MON 87429 grain was 2.4 mg/g DW.

The mean PAT (pat) protein level in MON 87429 across all sites was highest in OSL 1 at 5.8 mg/g DW and lowest in forage-root at 0.81 mg/g DW. The mean PAT (pat) protein level in MON 87429 grain was 0.84 mg/g DW.

The mean FT_T protein level in MON 87429 across all sites was highest in OSL1 at 440 mg/g DW and lowest in forage-root at 22 mg/g. The mean FT_T protein level in MON 87429 grain was 47 mg/g DW.

The mean CP4 EPSPS protein level in MON 87429 across all sites was highest in OSL 1 at 54 mg/g DW and lowest in grain at 0.63 mg/g DW.

5. Dietary Exposure

It is expected that MON 87429 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 87429.

6. Nutrition

To evaluate if there were any unanticipated consequences of the genetic modifications to MON 87429 maize, the nutritional and anti-nutritional components of the MON 87429 maize were analyzed and compared to a conventional control with a similar genetic background. This was done as part of a field trial conducted in 2017 with a randomized block design with four replicates at each of 5 field states in the United States. These sites included: Audubon County, Iowa; Vermilion County, Illinois; Boone County, Indiana; York County, Nebraska; and Miami County, Ohio. While there were no Canadian sites, the petitioner notes that these sites were located at similar latitudes and conditions to the maize-growing regions of Canada.

The compositional analytes measured in MON 87429 maize and non-genetically modified control maize grain were: protein, fat, total dietary fibre, ash, minerals (calcium, copper, iron, magnesium, manganese, phosphorus, potassium, sodium, and zinc), vitamins (B1, B2, B3, B6, B9, and vitamin E), beta-carotene, fatty acids (22 fatty acids), amino acids (18 amino acids), p-coumaric acid, furfural, ferulic acid, phytic acid, and raffinose.

Of the analytes measured, the following showed a statistically significant treatment effect in the MON 87429 maize compared to the control: total fat (lower in the MON 87429 maize than the control), palmitoleic acid (higher), stearic acid (higher), oleic acid (lower), linoleic acid (higher), linolenic acid (higher), behenic acid (higher), copper (lower), iron (lower), magnesium (lower), and vitamin E (lower). These differences between the MON 87429 maize and the control were acceptable as the analyte levels remained within OECD ranges. Palmitoleic acid and behenic acid (analytes not listed by OECD) each represent <0.2 % of the total fatty acids in both MON 87429 and control, and the differences are not of nutritional concern. The remaining analytes did not show statistically significant differences between MON 87429 and the control. The petitioner has demonstrated that MON 87429 maize has similar nutritional composition to its control.

Based on the information provided on the composition of MON 87429 and control LH244 varieties, there are no safety concerns with the use of MON 87429 as a food ingredient in Canada from a nutritional perspective.

7. Chemistry/Toxicology

The petitioner provided a scientific rationale for the lack of toxicity of all four proteins based on the lack of exposure to the proteins and the lack of similarity of the proteins to known toxins, as demonstrated by in vitro heat stability and digestion assays and in silico amino acid sequence homology analyses. For the FT_T protein, an acute mouse study was also submitted.

DMO, PAT (pat), and CP4 EPSPS proteins equivalent to those in MON 87429 were previously assessed and authorized by Health Canada for food use. DMO and PAT (pat) proteins from cottonseed MON 88701, equivalent to those proteins in previous maize line MON 87419, assessed in 2016, and this maize line MON 87429, were used in the heat stability and digestion studies provided. CP4 EPSPS protein from Sugar Beet Event H7-1, assessed in 2005, equivalent to this protein in MON 87429, was used in the heat stability and digestion studies provided.

The herbicide tolerant traits of maize MON 87429 are not expected to change the pattern of maize consumption in Canada. Most maize products would be expected to be heated to 75 degrees for 15 minutes during processing or cooking. Under these conditions, the four novel proteins are deactivated, based on the results of heat stability assays submitted. In general, deactivated proteins are expected to have a lower probability of causing negative effects on health compared to proteins with enzymatic activity.

The DMO, PAT (pat), FT_T, and CP4 EPSPS proteins were digested after 0.5 minutes in a simulated gastric fluid assay. The FT_T protein showed a persistent peptide in the gastric digestion assay, however the FT_T protein was digested completely after 2 minutes in simulated gastric fluid (pepsin) followed by 0.5 minutes in simulated intestinal fluid (pancreatin). Based on these in vitro digestion assays, the novel proteins are expected to be digested quickly and completely and therefore have little or no opportunity to negatively affect human health. The FT_T protein showed no acute oral toxicity in mice at the highest dose of 2000 mg/kg.

The petitioner compared the amino acid sequences of the novel proteins to those of known toxins using the GenBank protein database (release 223; 75 261 615 sequences) and keywords related to toxicity. No alignments were found for DMO, FT_T, or CP4 EPSPS proteins. The PAT (pat) protein showed weak alignment (43 % over 161 amino acids) to the toxin part of a toxin/anti-toxin system in bacteria however this system is only relevant to bacteria and therefore would not affect human health. Overall, no amino acid sequence homology was identified between these proteins and known toxins relevant to human health

The host organism, maize (Zea mays L.), has a long history of safe food use in Canada and is not associated with allergenic concerns. The petitioner noted that the 4 donor organisms are widespread in the environment and have not been reported to be sources of known allergens.

As noted above, the novel proteins are not expected to last long enough in the digestive tract to cause effects. This would include allergenicity, due to their inactivation by heat and quick and complete digestion, based on the results from in vitro heat stability and digestion assays submitted.

The petitioner conducted a bioinformatics analysis using the amino acid sequences of the 4 proteins, which were compared with sequences of known allergens using the COMPARE database (year 2018; 2038 sequences). No matches were identified. These proteins do not share sequence similarity with known allergens.

Based on the information reviewed, there are no safety concerns regarding the food use of MON 87429 compared to conventional maize varieties from a toxicological or allergenic perspective

Conclusion

Health Canada’s review of the information presented in support of the food use of MON 87429 does not raise concerns related to food safety. Health Canada is of the opinion that food derived from this maize variety is as safe and nutritious as food derived from current commercial maize varieties.

Health Canada's opinion deals only with the food use of MON 87429. 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.

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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