Novel food information: Insect protected cotton – MON 89151

Background

Health Canada has notified Bayer CropScience Inc. (Bayer) that it has no objection to the food use of insect protected cotton – MON 89151 (MON 89151).

Health Canada conducted a comprehensive assessment of this modified cotton 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 conducted by Health Canada. This document contains no confidential business information.

Introduction

Bayer has developed a novel cotton (Gossypium hirsutum) variety, MON 89151 that which exhibits resistance toward lepidopteran pests in countries including the United States (U.S.), Australia, and Brazil.

MON 89151 was developed through the introduction of three gene expression cassettes for the expression of a Cry1Da_7 gene encoding the insecticidal Cry1Da_7 protein, a Cry1B.3 gene encoding the insecticidal Cry1B.3 protein, and a Vip3Cb1.1 gene encoding the insecticidal Vip3Cb1.1 protein. Expression of the Cry1Da_7, Cry1B.3, and Vip3Cb1.1 proteins confer resistance towards lepidopteran pests. All three insecticidal proteins follow the same general mode of action in that they are ingested by the target organism as protoxins before being converted into their active toxin form in the insect midgut via proteolytic activation. The active form of the proteins bind to specific receptors in the insect midgut resulting in oligomerization at the membrane interface, leading to membrane pore formation in the midgut cells, which in turn leads to insect death.

The safety assessment performed by Food and Nutrition Directorate (FND) 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 reflects international guidance documents in this area (e.g., Codex Alimentarius). The assessment considered: how MON 89151 was developed, how the composition and nutritional safety of this variety compared to its conventional 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 FND 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 89151 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

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

Development of the Modified Plant

MON 89151 was created through Agrobacterium-mediated transformation of meristem tissues from cotton variety DP393 with plasmid PV-GHIR529967. The T-DNA region of the plasmid was integrated into the cotton genome and contained three gene expression cassettes (Cry1Da_7, Cry1B.3, and Vip3Cb1.1 genes) which confer to MON 89151 resistance to lepidopteran pests.

Characterization of the Modified Plant

The molecular characterization of MON 89151 was conducted using a combination of next-generation sequencing (NGS) and direct sequencing to determine the insert copy number and sequence of the T-DNA insert and the adjacent flanking genomic DNA.

Genomic DNA from MON 89151 and the DP393 parental line (control) were sequenced using NGS. The sequencing reads from MON 89151 and DP393 were each mapped against two reference sequences: (1) a single copy locus from the native plant G. hirsutum genome, region 70,825,501-70,828,501 (NC-053432.1), to determine sequencing depth, and (2) the transformation plasmid PV-GHIR529967 to characterize the intended T-DNA insert in MON 89151. A depth of coverage of greater than 75× was confirmed for each sample, providing comprehensive coverage and detection of all inserted DNA. Furthermore, the level of sensitivity achieved for the NGS was sufficient to detect 100 % of a spiked-plasmid sequence when present at both a 1-genome equivalent and a 1/10^th of a copy per genome equivalent.

Results of the sequencing and analysis indicated that a single copy of the intended T-DNA insert integrated into the genome of MON 89151 at a specific locus on chromosome D4. The T-DNA insertion does not disrupt any endogenous genes in the cotton genome. The three expression cassettes of the T-DNA insert have their intended sequence and organization, with the exception of small deletions in the right border (RB) and left border (LB) sequences of the T-DNA insert. No backbone sequences of the transformation plasmid are present in the MON 89151 genome.

Bioinformatics analyses were performed to ensure that any putative peptides encoded by the 5′ and 3′ inserted junction sequences of the MON 89151 T-DNA insert are not similar to allergenic, toxic, or potentially biologically active proteins that are known to cause adverse health effects. Peptide sequences spanning the 5′ and 3′ junction sequences were translated from stop-to-stop codon in all six reading frames. These putative peptide sequences were used as queries for FASTA and sliding window searches against the AD_2024 database, and FASTA searches against the TOX_2024 and PRT_2024 databases. No biologically relevant sequence similarities were observed between the translated putative junction sequences and allergens, toxins, or biologically active proteins associated with adverse effects for human or animal health.

The genetic stability of the T-DNA insert in MON 89151 was assessed by NGS. Genomic DNA from five generations of MON 89151 (R2, R3, R4, R5, and R6) were used. Genomic DNA from the parental line served as a negative control, with control genomic DNA spiked with plasmid PV-GHIR529967 serving as a positive control. The results showed that the single copy of the T-DNA insert is stably maintained across multiple generations of MON 89151.

The pattern of inheritance of the T-DNA insert in MON 89151 was demonstrated through segregation analysis of three generations (R2F2, R2F3, and R2F4). A Pearsons's chi-square (χ²) analysis was used to compare the observed segregation ratios of the MON 89151 T-DNA insert to the expected ratios for three segregation generations. The χ² value in the R2F2, R2F3, and R2F4 generations indicated no statistically significant difference between the observed and expected segregation ratios of MON 89151 T-DNA. These results support the conclusion that the MON 89151 T-DNA insert resides at a single locus within the cotton genome and is inherited according to Mendelian principles of inheritance.

Based on the available data provided, the Bureau of Microbial and Toxicological Food Safety has no safety concerns regarding MON 89151 from a molecular perspective.

Product Information

MON 89151 differs from its conventional counterpart by the expression of three insecticidal proteins: Cry1Da_7, Cry1B.3, and Vip3Cb1.1.

The Cry1Da_7 protein, encoded by the Cry1Da_7 gene, is a modified Cry1Da protein derived from the soil bacterium Bacillus thuringiensis. The Cry1B.3 protein, encoded by the Cry1B.3 gene, is a chimeric protein comprised of domains I and II from Cry1Be, domain III from Cry1Ka2, and a protoxin domain from Cry1Be, all derived from B. thuringiensis. The Vip3Cb1.1 protein, encoded by the Vip3Cb1.1 gene, is derived from a Paenibacillus bacterium in the Paenibacillus popilliae-containing clade.

All three proteins follow the same general mode of action in that they are ingested by the target organism as protoxins before being converted into their active toxin form in the insect midgut via proteolytic activation. The active form of the proteins bind to specific receptors in the insect midgut resulting in oligomerization at the membrane interface, leading to membrane pore formation in the midgut cells, which in turn leads to insect death. These novel expression products have not been previously assessed by Health Canada.

Expression of the Cry1Da_7, Cry1B.3, and Vip3Cb1.1 proteins in MON 89151 was analyzed in various tissues including leaf [over season leaf (OSL)1 and OSL4], square, pollen, root (OSR3), boll, and seed tissues. Plants were grown at five sites in the United States during the 2023 growing season. Expression levels of each protein were determined by validated enzyme-linked immunosorbent assay (ELISA).

The mean Cry1Da_7 protein level in MON 89151 across all sites was the highest in OSL1 at 180 µg/g dry weight (dw) and lowest in boll at below the Limit of Quantification (<LOQ). The mean Cry1Da_7 protein level in MON 89151 seed was 17 µg/g dry weight dw.

The mean Cry1B.3 protein level in MON 89151 across all sites was the highest in OSL1 at 510 µg/g dw and lowest in boll and root at <LOQ. The mean Cry1B.3 protein level in MON 89151 seed was 35 µg/g dw.

The mean Vip3Cb1.1 protein level in MON 89151 across all sites was the highest in OSL1 at 30 µg/g dw and lowest in boll at <LOQ. The mean Vip3Cb1.1 protein level in MON 89151 seed was 0.85 µg/g dw.

Dietary exposure

It is expected that MON 89151 will be used in applications similar to conventional cotton varieties. The petitioner does not anticipate a significant change in the food use of cotton with the introduction of MON 89151.

Nutrition

The petitioner provided compositional data for MON 89151 and the genetically similar conventional control DP393 obtained from cotton samples harvested from five field trials conducted in the U.S. during the 2023 growing season. Each field trial was planted with four replicates of each of the modified and conventional cotton in a randomized complete block design.

Samples were analyzed using acceptable methods for proximates, amino acids, fatty acids, carbohydrates by calculation, fibre, minerals, vitamin E, and anti-nutrients. The data provided was for all key nutrients and anti-nutrients as described in the Organisation for Economic Co-operation and Development (OECD) "Consensus Document on Compositional Considerations for New Varieties of Cotton (Gossypium hirsutum and Gossypium barbadense): Key food and feed nutrients and anti-nutrients" (2009).

Where a statistically significant difference (p-value <0.05) was identified between the modified cotton and its conventional control, the nutritional relevance of the difference was determined through comparison to the control range value (maximum value minus the minimum value) of the conventional control and the natural variability defined by the range of values observed in the literature and reported in the Agriculture and Food Systems Institute's Crop Composition Database for cotton.

There were no statistically significant differences between MON 89151 cotton and the conventional control for 30 of the 40 components analyzed. For the ten components (protein, tryptophan, myristic acid, palmitic acid, palmitoleic acid, oleic acid, carbohydrates by calculation, malvalic acid, sterculic acid, and dihydrosterculic acid) that showed statistically significant mean differences, between MON 89151 and the conventional control were less than the corresponding control range values, suggesting that the genetic modification does not impact levels of these components more than the natural variation observed within the conventional control grown at multiple locations.

The Bureau of Nutritional Sciences (BNS) has not identified any nutritional concerns related to food use of MON 89151 cotton.

Chemistry

Total and free gossypol content were included as part of the compositional comparative assessment for MON 89151 and were considered as part of the review conducted by the Bureau of Nutritional Sciences (BNS). Otherwise, chemical contaminant residue data have not been provided, nor have any unique contaminant considerations identified with respect to MON 89151. Cottonseed flour sold in Canada is required to comply with Health Canada's maximum level (ML) of 450 ppm free gossypol, which is set out in Part 2 of the List of Contaminants and Other Adulterating Substances.

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

The Pre-Market Toxicology Assessment Section (PTAS) performed a safety assessment of MON 89151, a cotton crop that expresses the proteins Cry1Da_7, Cry1B.3, and Vip3Cb1.1. The petitioner submitted studies to support the safety of each of these proteins. Studies were conducted in accordance with Good Laboratory Practices (GLP) and considered acceptable for the purpose of this safety assessment.

Surrogate proteins produced in separate bacterial expression systems were used to conduct the supporting safety studies (heat stability, in vitro digestion, and acute toxicity assays) for the three proteins. The Cry1Da_7 and Cry1B.3 proteins evaluated in these safety studies were produced from a B. thuringiensis (Bt) expression system and Vip3Cb1.1. protein was produced from an E. coli expression system. The petitioner provided information to confirm the equivalence of the microbial-derived proteins with the novel proteins present in the cotton. This included comparison of molecular weight, immunoreactivity, lack of glycosylation, amino acid sequence, and functional activity.

No acute oral toxicity of Bt-produced Cry1Da_7 and Cry1B.1 proteins and E. coli-derived Vip3Cb1.1 protein was observed in the mouse studies submitted. In each study, mice (7 to 10/sex/group), were exposed by gavage to 0 or 5000 mg/kg body weight (bw) Cry1Da_7, Cry1B.3 or Vip3Cb1.1 protein. The no observed adverse effect level (NOAEL) was ≥5000 mg/kg bw in all three studies.

Results of in vitro digestion assays (simulated gastric fluid with pepsin and simulated intestinal fluid with pancreatin) demonstrated that full-length Cry1Da_7, Cry1B.3 and Vip3Cb1.1 proteins are rapidly digested by pepsin or pancreatin alone or pepsin followed by pancreatin. Based on these findings, Cry1Da_7, Cry1B.3 and Vip3Cb1.1 proteins are highly unlikely to exert a systemic toxic effect to human health.

The results of heat stability assays showed that thermal treatment at 55 °C for 15 or 30 minutes inactivated the biological function of the Vip3Cb1.1 protein, while heat treatment at 75 °C for 15 minutes inactivated the biological function of Cry1Da_7 and Cry1B.3 proteins. This data suggests that these novel proteins would be inactivated when exposed to elevated temperatures during refined, bleached, and deodorized (RBD) oil processing or subsequent cooking. These findings suggest that consumers would not be exposed to any active proteins.

The amino acid sequences of Cry1Da_7, Cry1B.3 and Vip3Cb1.1 proteins were evaluated using FASTA bioinformatic alignment searches performed with 7,338 sequences in the TOX_2024 database (last updated 2024); a subset of sequences derived from the Swiss-Prot database using keyword-specific exclusion criteria to retain relevant toxins. There were no relevant similarities between Cry1Da_7, Cry1B.3 or Vip3Cb1.1 proteins and known toxins that are harmful to humans.

Dietary exposure to cottonseed is primarily limited to refined, bleached, and deodorized (RBD) oil and cottonseed linters used as a source of food grade cellulose. Both products are highly processed and expected to contain very low amounts of protein. Results from field expression studies provided by the petitioner showed that cotton seeds contain the lowest level of these novel proteins (Cry1Da_7, Cry1B.3 and Vip3Cb1.1) compared to other parts of the cotton plant. Therefore, the dietary exposure to the Cry1Da_7, Cry1B.3 and Vip3Cb1.1 proteins from MON 89151 was considered negligible.

Based on the available information, the PTAS has not identified any toxicological safety concerns with the proposed food use of MON 89151.

Allergenicity

MON 89151 produces three novel insecticidal proteins, Cry1Da_7, Cry1B.3, and Vip3Cb1.1, which protect against feeding damage caused by targeted lepidopteran insect pests.

The donor organisms B. thuringiensis and Paenibacillus sp. from Paenibacillus popilliae-containing clade, are widespread in the environment and have not been reported as a source of known allergens.

Cottonseed and cottonseed meal have a long history of safe food use in Canada and is not associated with allergenic concerns. Furthermore, human dietary exposure to cottonseed is limited to cottonseed oil and cotton linters that contain negligible protein.

To assess the allergenic safety of the Cry1Da_7, Cry1B.3 and Vip3Cb1.1 proteins their amino acid sequences were compared to the sequences in known allergens, gliadins, and glutenins in the COMprehensive Protein Allergen Resource (COMPARE) database. The database was searched using a matching criteria of ≥35 % over a sliding window of 80-amino acid sequences, as well as a matching criteria of 100 % over 8-amino acids, both criteria being consistent with guidance from WHO/FAO.

Based on the search criteria no matches were identified. No significant homology between the insecticidal proteins in MON 89151 and known or suspected food allergens were identified, using international criteria (Codex Alimentarius).

Thermal stability and in vitro digestive fate experiments were conducted with Cry1Da_7, Cry1B.3, and Vip3Cb1.1 proteins which demonstrated that these proteins are expected to be rapidly digested by pepsin and pancreatin, and degraded at temperatures below those of cottonseed oil and cotton linters manufacturing.

Based on the available information, the PTAS did not identify any food allergy safety concerns related to the proposed use of MON 89151.

Conclusion

Health Canada's review of the information presented in support of the use of MON 89151 does not raise concerns related to food safety.

Health Canada's opinion refers only to the food use of MON 89151. 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 and Nutrition 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
bmtfs.inquiries-requetes.bsamt@hc-sc.gc.ca

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2026-09-14