Novel Food Information: Herbicide Tolerant and Pest Resistant Maize line 4114

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. Toxicology
  8. Allergenicity
• Conclusion

Background

Health Canada has notified Pioneer Hi-Bred Production LP that it has no objection to the food use of herbicide tolerant and pest resistant maize line 4114. 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 Pioneer Hi-Bred Production LP and the evaluation by Heath Canada and contains no confidential business information.

1. Introduction

Maize event 4114 was genetically modified using recombinant DNA techniques in order to confer resistance to lepidopteran and coleopteran insect pests through the expression of the Cry1F, Cry34Ab1, and Cry35Ab1, insecticidal proteins, and tolerance to glufosinate herbicides through the expression of the phosphinothricin N-acetyltransferase (PAT) protein. The cry1F gene is from Bacillus thuringiensis var aizawai, the cry34Ab1 and cry35Ab1 genes from B. thuringiensis strain PS149B1, and the pat gene from Streptomyces viridochromogenes. Maize event 4114 is a molecular stack of two previously approved lines (1507 and 59122).

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 harmonized efforts with other regulatory authorities and reflect international guidance documents in this area (eg. Codex Alimentarius). The assessment considered: how maize event 4114 was developed; how the composition and nutritional quality of maize event 4114 compared to non-modified maize varieties; and what the potential is for maize event 4114 to be toxic or cause allergic reactions. Pioneer has provided data which demonstrates that maize event 4114 is as safe as traditional 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 (Novel Foods). Foods derived from maize line 4114 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

• (i) 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 maize event 4114 and data that characterizes the genetic modification that confers insect resistance and herbicide tolerance through the expression of the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins. Maize event 4114 was produced via Agrobacterium-mediated transformation of PHWWE maize with the vector PHP27118.

The transformation vector contains a single multi-gene transfer DNA (T-DNA) consisting of the cry1F, cry34Ab1, cry35Ab1, and pat gene cassettes. The T-DNA contains four expression cassettes which are composed of all the same elements (promoters, protein coding sequences, and terminators) as those found in the previously assessed maize lines 1507 (Cry1F, PAT) and 59122 (Cry34Ab1, Cry35Ab1, PAT).

The first expression cassette contains a truncated version of the cry1F gene from Bacillus thuringiensis var aizawai which is under the control of the maize polyubiquitin promoter gene ubiZM1 which results in the constitutive expression of the Cry1F protein in maize. The transcription termination sequence for the cry1F gene is the polyadenylation signal from open reading frame 25 of the A. tumefaciens Ti plasmid pTi15955.

The second expression cassette contains the cry34Ab1 gene isolated from B. thuringiensis strain PS149B1 which is under the control of a second copy of the maize polyubiquitin promoter with 5’ UTR and intron. The transcription termination sequence for the cry34Ab1 gene is the 3’ terminator sequence from the proteinase inhibitor II (pinII) gene of Solanum tuberosum.

The third expression cassette contains the cry35Ab1 gene also isolated from B. thuringiensis strain PS149B1 which is under the control of the Triticum aestivum (wheat) peroxidase promoter and leader sequence. The transcription termination sequence for the cry35Ab1 gene is a second copy of the pinII terminator.

The fourth expression cassette contains a version of the phosphinothricin N-acetyltransferase (pat) gene from Streptomyces viridochromogenes that has been optimized for expression in maize and which is under the control of the promoter and terminator regions from the cauliflower mosaic virus (CaMV) 35S transcript.

3. Characterization of the Modified Plant

Southern blot analysis was used to determine the number of copies and insertion sites of the integrated T-DNA as well as the presence or absence of plasmid backbone sequence. This analysis demonstrated a single copy of the T-DNA inserted at a single site and at a single locus of the maize genome. Maize line 4114 genomic DNA probed for backbone sequences showed no detectable hybridization bands, thus maize line 4114 does not contain backbone sequences from the transformation vector PHP27118.

In addition to Southern blot analysis, genomic DNA extracted from maize line 4114 was subjected to DNA sequencing analysis. Sequencing of the insert and the flanking genomic DNA confirmed the arrangement and linkage of the various elements within the insert except for a 29 base pair deletion at the Right Border end of the insert and a 24 base pair deletion at the left border end of the insert, which is not uncommon for Agrobacterium- mediated transformation events.

An analysis of putative polypeptides encoded by DNA spanning the 5’and 3’ genomic DNA junctions was performed using a bioinformatic comparison strategy to evaluate the potential for novel protein open reading frames (ORFs) that may have homology to known toxins or allergens. A single potential ORF was identified, but this ORF was shown to lack the regulatory elements necessary for transcription. In the unlikely event that a translation product was derived from DNA spanning the potential ORF, the putative polypeptide would lack any sequence similarity with known allergens or toxins.

Southern blot analysis was performed using four generations of maize line 4114. The digested genomic DNA showed the expected banding pattern across all samples from each of the four generations tested, showing the stability of maize line 4114 at the genomic level. Segregation data further confirmed the stability of the insert, showing that it segregates according to Mendelian inheritance principles. These results were also consistent with the molecular characterization data that indicated a single genomic insertion site.

4. Product Information

Four new proteins are expected to be expressed in maize line 4114 based on the characterization of the inserted genetic material. The cry1F gene expressed in maize line 4114 encodes a 68 kDa Cry1F protein, the cry34Ab1 gene encodes a 14 kDa protein, the cry35Ab1 gene encodes a 44 kDa protein, and the pat gene encodes a 21 kDa protein. The petitioner explained that these same proteins are found in the commercial hybrid Herculex® XTRA which is the conventional cross 1507x59122.

Characterization of the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins in maize line 4114 was conducted using sequencing and Western blot analysis. The amino acid sequences of the four proteins were determined from the nucleotide sequences of the corresponding genes present in plasmid PHP27118. In each case, the amino acid sequences of the proteins inserted in maize line 4114 were identical to the amino acid sequences found in plasmid PHP27118, and identical to the deduced amino acid sequences of the respective proteins expressed in either 1507 or 59122 maize lines. Western blot analysis comparing the molecular weight and immunoreactivity of each protein isolated from 4114 maize and 1507x59122 demonstrated that the respective proteins were equivalent. These analyses demonstrate that the plant produced proteins in maize line 4114 are equivalent to the plant produced proteins in lines 1507 and 59122, which were previously found to be equivalent to the microbe produced proteins that were used in the previously assessed toxicology studies.

The expression levels of the four proteins in maize grain, the source of all maize derived food products, were assessed using ELISA. For each protein, the concentration in grain samples from maize line 4114 was compared to concentrations in grain samples from 1507, 59122, and 1507 x 59122. The mean values were 3.3 ug/g tissue dry weight for Cry1F, 24 ug/g tissue dry weight for Cry34Ab1, 1.1 ug/g tissue dry weight for Cry35Ab1, and below the limit of quantification (0.069 ug/g tissue dry weight) for PAT. The levels of the four proteins in maize line 4114 were similar to corresponding values measured in samples of 1507, 59122, and 1507 x 59122 maize.

5. Dietary Exposure

The genetic modification of maize line 4114 is not intended to alter any of its nutritional aspects. Therefore, the use of maize line 4114 and products derived from it will be similar to traditional maize varieties. The introduction of maize line 4114 will only replace a fraction of current maize varieties and is not anticipated to result in a change in the dietary intake of maize and maize-derived products.

6. Nutrition

A crop composition study with maize line 4114 and a near-isogenic control grown in side-by-side trials in 2010 at six locations within the commercial maize growing regions of the United States and Canada, with four sites in the United States (Richland, IA; Wyoming, IL; Geneva, MN; and York, NE) and two sites in Canada (Branchton, ON; and Thorndale, ON) was examined. Each field site utilized a randomized complete block design with maize line 4114 and the control planted in two-row plots within four replicate blocks.  Samples for compositional analysis were collected from plots that did not receive a glufosinate herbicide treatment. However, irrigation, fertilizers and maintenance chemicals were uniformly applied to all plots at each trial site location in order to ensure production of a successful crop. In two separate experiments, using methods similar to those employed in the composition study previously described, grain samples were harvested from trials of non-modified commercial reference hybrids grown at six locations in 2003 and in 2007 and used to derive a tolerance interval for comparison purposes for the current study.

The compositional analytes measured in maize line 4114 and the control grain were: proximate content (ash, crude fat, crude protein, carbohydrate), fiber (ADF, NDF, crude fiber), minerals (calcium, copper, iron, magnesium, manganese, phosphorus, potassium, sodium, zinc), vitamins (β-carotene, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B9, α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, and total tocopherol), amino acids (aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, arginine, tryptophan), fatty acids (palmitic, stearic, oleic, linoleic, α-linolenic,  arachidic, eicosenoic, lignoceric, caprylic, capric, lauric, myristic, myristoleic, pentadecanoic, pentadecenoic, palmitoleic, heptadecanoic, heptadecenoic, heptadecadienoic, (9,15) isomer of linoleic, γ-linolenic, nonadecanoic, eicosadienoic, eicosatrienoic, arachidonic, heneicosanoic, behenic, erucic, and tricosanoic), secondary metabolites and anti-nutrients (ρ-coumaric acid, furfural, ferulic acid, phytic acid, inositol, raffinose, trypsin inhibitor).

Results indicated that a few of the analytes (6 of 82) in maize line 4114 showed a statistically significant difference compared to the control. However, these differences were considered acceptable because, in all cases, the level of the analyte in maize line 4114 was within the literature ranges reported in the Organisation for Economic Co-operation and Development Consensus Document on Compositional Considerations for New Varieties of Maize (when available) and the International Life Sciences Institute Crop Composition Database, and within the tolerance intervals included in the analysis.

Statistical analysis was not conducted on several analytes (26 of 82) in maize line 4114 since >80% of samples’ levels were less than the LOQ. The levels of these analytes were acceptable for one of the following reasons: The level of the analyte in maize line 4114 was within the tolerance interval included in the analysis; or the level of the analyte in maize line 4114 would not impact dietary intakes amongst the general population or pose a nutritional safety concern.

7. Toxicology

Health Canada assessed information provided on the potential toxicity of maize line 4114. This included information on the potential toxicity of the novel proteins produced by maize line 4114 (Cry1F, Cry34Ab1, Cry 35Ab1 and PAT proteins), as well as the potential toxicity of the whole food (corn).

Health Canada has previously approved the novel expression of S. viridochromogenes PAT protein in soybean, cotton, sugarbeet, canola and maize lines intended for human consumption.  The safety of PAT protein is well established and as such, the consumption of PAT protein from maize line 4114 is not expected to pose a toxicological human health concern.

Health Canada has also previously approved genetically modified maize lines producing the Cry1F, Cry34Ab1, Cry35Ab1 and PAT proteins (e.g. 1507 produces Cry1F and PAT; 59122 produces Cry34Ab1, Cry35Ab1 and PAT). No adverse reactions have been reported in Canada or other countries, which have approved these maize lines.  Additionally, no adverse reactions have been reported for the commercial hybrid, Herculex® XTRA that was generated by breeding of maize lines 1507 and 59122 and contains the same transgenic proteins (Cry1F, Cry34Ab1, Cry35Ab1 and PAT) as maize 4114. This information supports the general safety of this product.

Cry1F, Cry34Ab1 and Cry35Ab1 did not produce any adverse effects when given as an acute oral dose to mice. These results demonstrate that ingestion of Cry1F, Cry34Ab1 and Cry35Ab1 proteins would not result in any adverse health effects at doses up to 576 mg, 2700 mg and 1850 mg/kg b.w., respectively (the highest doses tested).

The petitioner provided the results of an in silico search which compared the amino acid sequences of Cry34Ab1 and Cry35Ab1 to amino acid sequences of known toxins. The results of the search determined that Cry34Ab1 and Cry35Ab1 did not share significant sequence similarity with any known toxins (other than other Cry proteins).

The petitioner provided the results of in vitro simulated gastric fluid assays, which demonstrated that microbially-derived Cry1F, Cry34Ab1 and Cry35Ab1 proteins were rapidly digested (within 1-20 minutes) and suggests that when these Cry proteins are ingested, they are unlikely to gain access to the general circulatory system and therefore unlikely to exert a systemic toxic effect.

The results of the heat stability assay showed that microbially-derived Cry34Ab1 and Cry35Ab1 were inactivated at temperatures commonly involved in corn processing (60-90 ºC). This finding suggests that consumers would not be exposed to active protein.

A 13-week repeated-dose oral toxicity study was performed with Sprague-Dawley rats (12 rats per sex per group; 6-8 weeks of age) fed grain from 4114 maize, 4114 maize sprayed with glufosinate ammonium, non-transgenic near-isogenic control maize, or commercially available non-transgenic hybrid reference maize (3 varieties tested) as a whole food which constituted 32 % w/w of the diet. This study was conducted in accordance with OECD guidelines for 90-day repeated dose oral toxicity studies in rodents. No treatment-related incidences of mortality or changes in clinical observations, body weight, food intake, clinical chemistry, hematology or urinalysis were reported.

However, at necropsy, two male rats of the maize 4114 treatment group were observed to have developed renal tumours, specifically amphophilic vacuolar renal tubule adenomas/adenocarcinomas. One male had multiple benign adenomas in each kidney, while the other male had a benign adenoma in each kidney and a single carcinoma in one kidney. Renal tumours were absent in all other treatment groups (rats given maize 4114 treated with glufosinate, isogenic control maize or commercial control maize). Other than the presence of the tumours, the affected animals showed no evidence of toxicity, i.e., all other measured parameters were similar to all other animals.

The authors concluded that the renal tubule adenomas/carcinomas were spontaneous and not a treatment-related effect based on the reports of histologically identical tumours in many rat strains, including Sprague Dawley rats (occurring as early as 12 weeks of age) in the previous literature and in publications from the supplier (Charles River). Typically, tumours that arise due to chemical treatment are late-onset as they require more time to develop. It is for this reason that chronic toxicity (2-year) studies are used to identify potential cancer hazards. The tumours reported in this study have a young age of onset and occur in the absence of nephrotoxicity and metastatic activity which suggests that they are spontaneous and of familial origin. No biologically relevant renal effects were observed in any other animal in this group and none were observed in rats given maize 4114 treated with glufosinate. This finding is consistent with the conclusion that the effect was not due to the presence of maize 4114 in the diet. The authors of the study concluded that maize line 4114 does not contain substances that are toxic at the levels provided in the whole-food feeding study. The authors’ conclusion that the observed tumours were not a treatment-related effect was supported by a published review from a panel consisting of five experts (Hardisty et al. 2013)^{Footnote 1}.

An in-house review by an expert from the Toxicology Research Division and the assessment performed by PTAS were of the opinion, consistent with the study’s original authors and the panel of experts, that the tumours observed in the maize 4114 fed rats were spontaneous in nature, and should be considered incidental and not treatment-related.

The expression level of Cry1F, Cry34Ab1, Cry35Ab1 and PAT proteins in 4114 maize grain is considered very low (0.069 – 24 µg protein/g maize grain d.w.) and the estimated intake in the subpopulation which consumes the greatest amount of corn products (on a w/w basis), children 6 years of age and younger (97.5th percentile), is expected to be very low (59.4, 432.2, 19.8 and 1.2 µg/kg b.w./day, respectively). The margin of exposure (MOE) between the predicted levels of exposure and the no-observable-effect level (NOEL) in the acute oral toxicity studies in mice were about 1 X 104, 6 X 103 and 1 X 105 for Cry1F, Cry34Ab1 and Cry35Ab1, respectively.  These MOEs are considered sufficiently large to account for any uncertainties in the toxicological database and exposure estimates.

Taken together, the small quantities of Cry1F, Cry34Ab1 and Cry35Ab1 proteins expressed in maize 4114 would not be expected to survive digestion and food processing to function as an active toxin in consumers. Additionally, no instances of toxicity have been reported by consumers since the approval of maize lines 1507 (Cry1F; NF-75) and 59122 (Cry34Ab1, Cry35Ab1; NF-122).

Maize line 4114 would not be expected to pose a toxicological health concern when consumed and is considered as safe as conventional maize currently available in the Canadian market.

8. Allergenicity

Health Canada assessed information provided on the potential allergenicity of maize line 4114 as a food. The information included reference to maize lines DAS-59122-7 (NF-122) and DAS-1507-1 (NF-75) which Health Canada had previously assessed and had no objection to their sale in Canada.

Health Canada has previously approved the novel expression of S. viridochromogenes PAT protein in soybean, cotton, sugarbeet, canola and maize lines intended for human consumption.  The safety of PAT protein is well established and as such, the consumption of PAT proteins from maize 4114 is not expected to pose an allergenic human health concern.

Health Canada has also previously approved genetically modified maize lines producing the Cry1F, Cry34Ab1, Cry35Ab1 and PAT proteins (e.g. 1507 produces Cry1F and PAT; 59122 produces Cry34Ab1, Cry35Ab1 and PAT). No allergenic reactions have been reported in Canada or other countries, which have approved these maize lines. No allergenic reactions have been reported for the commercial hybrid Herculex® XTRA that was generated by breeding maize 1507 with 59122 and contains the same transgenic proteins (Cry1F, Cry34Ab1, Cry35Ab1 and PAT) as maize 4114.

The in silico search compared the amino acid sequences of Cry1F, Cry34Ab1 and Cry35Ab1 to amino acid sequences of putative or known allergens. The results of the search determined that Cry1F, Cry34Ab1 and Cry35Ab1 proteins were not found to share significant sequence homology with putative or known allergens.

The results of the in vitro simulated gastric fluid assays demonstrated that microbially-derived Cry1F, Cry34Ab1 and Cry35Ab1 proteins were rapidly digested (within 1-20 minutes) and suggests that when these Cry proteins are ingested, they are unlikely to gain access to the general circulatory system and therefore unlikely to exert an allergenic effect.

The results of the heat stability assay showed that microbially-derived Cry34Ab1 and Cry35Ab1 were inactivated at temperatures commonly involved in corn processing (60-90 ºC). This finding suggests that consumers would not be exposed to active protein.

The expression level of Cry1F, Cry34Ab1, Cry35Ab1 and PAT proteins in 4114 maize grain is considered very low (0.069 – 24 µg protein/g maize grain d.w.) and the estimated intake in the subpopulation which consumes the greatest amount of corn products (on a w/w basis), children 6 years of age and younger (97.5th percentile), is expected to be very low (59.4, 432.2, 19.8 and 1.2 µg/kg b.w./day, respectively).

Taken together, it is unlikely that the small quantities of Cry1F, Cry34Ab1 and Cry35Ab1 proteins expressed in 4114 would survive digestion and food processing to function as an allergen. Additionally, no instances of allergenicity have been reported by consumers since the approval of maize 1507 and 59122 (NF-75 and NF-122 respectively).

Maize line 4114 would not be expected to pose an allergenic health concern when consumed.

Conclusion

Health Canada’s review of the information presented in support of the food use of herbicide tolerant and pest resistant maize line 4114 does not raise concerns related to food safety.

Health Canada's opinion deals only with the food use of maize line 4114.

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
bmh-bdm@hc-sc.gc.ca