Novel food information: Purple Tomato – Del/Ros1-N event

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 Norfolk Healthy Produce (hereafter, Norfolk) that it has no objection to the food use of Purple Tomato – Del/Ros1-N event (hereafter, Purple Tomato). Health Canada conducted a comprehensive assessment of this tomato 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 safety of Purple Tomato for food use was assessed as part of the Health Canada-Food Standards Australia New Zealand (FSANZ) shared assessment process. For this product, Health Canada was the primary assessor and conducted the safety assessment of this tomato line. FSANZ acted as the secondary assessor, peer-reviewing Health Canada's initial assessment report for Purple Tomato and providing feedback regarding the report.

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

Introduction

A novel food submission was received from Norfolk on September 10, 2024, regarding the acceptability for food use of a tomato that has been genetically modified to produce more anthocyanins in the fruit, resulting in a purple-coloured fruit. To create the Purple Tomato, Agrobacterium-mediated transformation was used to introduce 3 gene cassettes to the tomato variety MicroTom.

The elevated anthocyanin levels and purple colour in the fruit of Purple Tomato are the result of the introduction of the Delila (Del) and Rosea1 (Ros1) genes. The Del and Ros1 genes encode transcription factors that regulate genes involved in anthocyanin biosynthesis and were derived from snapdragon (Antirrhinum majus).

The Purple Tomato also contains the neomycin phosphotransferase II (nptII) gene which confers kanamycin resistance to the plant and was used as a selectable marker. The nptII gene is derived from Escherichia coli Bacterial transposon Tn5 and has been assessed in other genetically modified crop events that have been previously reviewed by Health Canada.

Purple Tomato was assessed under the Health Canada and Food Standards Australia New Zealand (FSANZ)'s Safety Assessment Sharing Initiative. For this assessment, Health Canada was the primary assessor and the safety assessment was conducted according to Health Canada's Guidelines for the Safety Assessment of Novel Foods. FSANZ evaluators peer reviewed the assessment conducted by Health Canada. The assessment considered: how Purple Tomato was developed, how the composition and nutritional safety of this variety compared to its conventional comparator, and the potential for this variety to present a toxic or allergenic concern. Norfolk has provided data to support that this variety is safe for use as food in Canada.

The Food and Nutrition 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 Purple Tomato 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

Purple Tomato was created through Agrobacterium-mediated transformation of leaf discs from tomato variety MicroTom with plasmid pDEL.ROS. The T-DNA region of the plasmid that was integrated into the tomato genome contained 3 gene expression cassettes which confer to Purple Tomato higher levels of anthocyanins in the fruit and resistance to kanamycin.

Purple Tomato expresses 2 novel proteins which confer the higher anthocyanin level trait: Del and Ros1, both derived from snapdragon (A. majus). Del is a basic-helix-loop-helix-type transcription factor and Ros1 is a Myb-type transcription factor. These transcription factors were chosen for their role in regulating genes involved in anthocyanin biosynthesis. The Del and Ros1 genes are controlled by tomato fruit-specific promoters in order to upregulate the tomato's native anthocyanin biosynthesis genes during fruit ripening, resulting in higher levels of anthocyanins in the fruit and the observed purple colour.

Purple Tomato also expresses the novel protein NPTII which confers kanamycin resistance and served as a selectable marker for transformation. The nptII gene is derived from the E. coli Bacterial transposon Tn5. The NPT II protein has been previously assessed in several other genetically modified crops by Health Canada, including Arctic^®apple events GD743 and GS784, corn event MON 87460, and Suppressed Polygalacturonase Activity Tomato Hybrids 1401F, H282F, 11013F, 7913F.

The petitioner provided information to support the safety and historical use of the source organisms: A. majus and E. coli. Based on the information provided, it was demonstrated that none of the source organisms are pathogenic.

Characterization of the modified plant

Data has been provided on a variety of generations in the Purple Tomato breeding pedigree. To ensure that the data presented on the various generations supported the safety of the intended commercial variety, the breeding pedigree was carefully assessed. The original T0 transformant in MicroTom was used to generate a T1 generation via self-pollination. This MicroTom T1 generation was then used to generate 6 self-pollinated MicroTom generations (T2-T6). The same MicroTom T1 generation plant was also crossbred into the MoneyMaker variety and the resulting MoneyMaker F1 generation was then self-pollinated for 9 generations (F2-F9). Further crossbreeding in the Goldkrone and VF36 varieties was also undertaken using the F9 MoneyMaker generation, which were likewise self-pollinated for multiple generations.

The results of the molecular characterization demonstrated that the introduced genetic changes in the Purple Tomato are equivalent in the T6 plant of the MicroTom line and the F9 plant of the MoneyMaker line. Based on this equivalence, the data presented from the various generations in both MicroTom and MoneyMaker were relevant and acceptable to demonstrate the safety of the intended commercial Purple Tomato.

The molecular characterization of Purple Tomato was conducted through a combination of Southern blot, polymerase chain reaction (PCR), quantitative PCR (qPCR), inverse PCR (iPCR), Sanger sequencing, and whole genome sequencing (WGS). Southern blot using the Ros1 cDNA as a probe was used to assess the number of insertions in Purple Tomato in the first generation (T0). qPCR was used to determine the insertion copy number through quantification of the number of nptII gene copies. iPCR was used to determine the genetic sequence of the regions flanking the borders of the inserted T-DNA. WGS was used to determine the sequence of the T-DNA insertion and assess for plasmid backbone sequences integrated into the Purple Tomato genome.

The results from the Southern blot, qPCR, and iPCR analyses indicated that two T-DNA insertions were present in the T0 Purple Tomato plant. The 2 insertions were determined to be on different chromosomes, and the insertion at locus A was segregated away during downstream breeding of Purple Tomato, leaving only the insertion at locus B. The loss of the insertion at locus A was confirmed by PCR and WGS analysis. The remainder of this document only discusses the T-DNA insertion at locus B.

The WGS analysis determined that the insertion at locus B contained a single copy of the intended T-DNA with the intended organization, except for small deletions of 52 bp from the right border and 75 bp from the left border. Border region deletions of a T-DNA insertion are common during Agrobacterium-mediated plant transformation due to DNA repair mechanisms. Given that the left and right borders are not part of the expression cassettes, it is considered unlikely to affect the function of the inserted genetic elements or the food safety of Purple Tomato.

The WGS analysis of the Purple Tomato demonstrated that there were no pDEL.ROS plasmid backbone sequences present in the genome of the Purple Tomato, including any antibiotic resistance genes.

To determine the location of the T-DNA insertion in the Purple Tomato genome, the identified tomato genomic sequences flanking the insertion site were subjected to homology search against a reference tomato genome (SL3.0 genome assembly, GCA_000188115.3). This search located the T-DNA insert on chromosome 4. The T-DNA insertion caused a 94 bp deletion of the tomato genome sequence but did not disrupt any endogenous genes or any other known annotated feature in the tomato genome.

Bioinformatics analyses were performed to assess the potential toxicity, allergenicity, or biological activity of putative peptides that may result from the T-DNA insertion. Potential open reading frames (ORFs) of ≥ 30 amino acids in length spanning the 5′ and 3′ insert-flank junctions of the Purple Tomato were translated in silico from the initiator codon to the stop codon in all six reading frames. No ORF sequences of greater than 30 amino acids were detected at the left border:T-DNA junction. A theoretical 32 amino acid peptide containing an initiating methionine codon and spanning the right border:T-DNA junction was identified. Norfolk indicates that there is no evidence that the sequence is translated into a functional protein as it lacks a nearby promoter or Kozak consensus sequence around the initiating ATG codon.

To assess potential toxicity of the putative peptide, a similarity search against NCBI non-redundant (nr) protein sequences was performed using the Basic Local Alignment Search Tool (BLAST; 2025; https://blast.ncbi.nlm.nih.gov/Blast.cgi). No alignments were returned between the putative peptide and any protein sequence in the NCBI database.

To assess potential allergenicity, a similarity search against the AllergenOnline database (2025; http://allergenonline.org/) was performed. A full-length sequence search using a FASTA (Fast Alignment Search Tool – All) alignment was performed comparing the predicted ORF sequence to the database entries. An 8-mer exact match FASTA search was performed comparing contiguous 8 amino acids within the ORF to the database entries. No alignments were returned between the putative peptide and any protein sequence in the AllergenOnline database.

The genetic stability of the T-DNA insertion in Purple Tomato was assessed by PCR to amplify the T-DNA:genomic junction sequences at the left and right borders followed by Sanger sequencing. Left border integrity was assessed in T1 and T6 plants from the MicroTom line. Right border integrity was assessed in a T6 plant from the MicroTom line and a F9 plant from the MoneyMaker line. PCR analysis was also performed on the left border region of the F9 plant of the MoneyMaker line. The results demonstrated that the inserted T-DNA is maintained stably across 5 generations in the MicroTom genetic background and has been stably maintained through crossing into the MoneyMaker background.

Further supporting the genetic stability of the T-DNA insertion, WGS analysis of a T6 plant from the MicroTom line and a F7 plant from the Goldkrone line demonstrated that the T-DNA insertion sequence was unchanged across multiple generations, including intraspecific crosses.

The pattern of inheritance of the T-DNA insertion in Purple Tomato was demonstrated through segregation analysis of Purple Tomato plants derived from crossing the homozygous F9 plant of the MoneyMaker with the VF36 tomato variety. Plants from the segregating F2 generation were evaluated by kanamycin resistance and fruit colour. A Chi-square test was used to compare the observed segregation ratio of the Purple Tomato to the expected ratio (3 kanamycin resistant/purple fruit : 1 kanamycin sensitive/red fruit). The results of the Chi-square analysis of the segregating progeny indicated no statistically significant difference between the observed and expected segregation ratios. These results support the conclusion that the T-DNA insert resides at a single locus within the Purple Tomato and is inherited according to Mendelian principles.

The phenotypic stability of the purple fruit trait in Purple Tomato was monitored by Norfolk over multiple generations and through crosses into other tomato varieties. Norfolk indicates that no variation in penetrance of the trait within a variety has been observed and that the purple fruit phenotype is stable.

Product information

Expression of the Del, Ros1, and NPTII proteins in Purple Tomato was assessed in the ripe fruit using liquid chromatography-mass spectrometry (LC-MS/MS). To establish the detection limit of this method, two standard proteins were spiked at different amounts into the Purple Tomato protein extract. The limit of detection was determined to be: 4 nanograms per gram fresh weight (ng/gfw) for Del, 1.5 ng/gfw for Ros1, and 1.7 ng/gfw for NPTII. The results indicated that the levels of Del, Ros1, and NPTII proteins were all below the limit of detection.

Expression of the Del and Ros1 mRNA transcripts were assessed in Purple Tomato fruit using northern blot analysis. The results indicated that the Del and Ros1 genes are expressed in Purple Tomato fruit.

Norfolk indicated that for the protein digestibility studies they expressed and purified the Del and Ros1 proteins as His-tagged fusion proteins in E. coli. As the Del and Ros1 proteins are expressed in the Purple Tomato below the limit of detection, it is not possible to do protein equivalency studies. Norfolk provided gel electrophoresis analysis of the E. coli-produced Del and Ros1 proteins which supports that the surrogate proteins are acceptable for use in digestion studies.

Based on the available data provided, Health Canada has no safety concerns regarding Purple Tomato from a molecular perspective.

Dietary exposure

The intended purpose of Purple Tomato is to provide a tomato with higher levels of anthocyanins. It is not expected by the petitioner that the introduction of Purple Tomato into the marketplace will result in a significant change in the food use of tomatoes.

Nutrition

The petitioner provided compositional data for Purple Tomato, and an isogenic non-genetically modified tomato (conventional control), collected from a glasshouse in the United Kingdom in 2018. The experiment included five plants each from the Purple Tomato and control variety, grown in controlled environmental conditions.

Fruits from both varieties were harvested at typical harvest maturity ("red-ripe"), at the same time, and analyzed using acceptable methods for proximates, vitamins, minerals, and carotenoids, as suggested by the Organization for Economic Co-Operation and Development (OECD) Consensus Document on Compositional Considerations for New Varieties of Tomato: Key Food and Feed Nutrients, Toxicants and Allergens.

For each analyte, statistical analyses were conducted to compare the composition of the Purple Tomato with the control variety. When statistically significant differences (P-value <0.05) between the control and the Purple Tomato were noted, the nutritional relevance of these differences was further examined by comparing the results to the expected range as per the natural variability for conventional tomatoes based on publicly available data sources, including the U.S. Department of Agriculture (USDA) Food Composition Database and scientific literature.

Statistically significant differences between the Purple Tomato and the conventional control were observed for 8 analytes: ash, glucose, total sugar, magnesium, potassium, folate, vitamin K, and lycopene. However, the Purple Tomato values were within the expected range for conventional tomato in all cases except vitamin K. The mean value of vitamin K in Purple Tomato was slightly lower than the reported range of values for conventional tomatoes. Given that vitamin K is present in a wide range of foods and the vitamin K content in Purple Tomato is minimally lower (0.2 µg/100 gfw) than the range reported in conventional tomatoes, consumption of Purple Tomato is not expected to meaningfully impact vitamin K intakes of consumers.

The petitioner also submitted information on the anthocyanin content of Purple Tomato (40-283 mg/100 gfw). Anthocyanins are present in Purple Tomato in the range found in commonly consumed foods (e.g., blackberry, blueberry, cherry, red grapes, eggplant, etc.) including other varieties of tomatoes (3.00-588.86 mg/100 gfw ^{Footnote 1}). In addition, the predominant anthocyanins present in Purple Tomato (i.e., Delphinidin 3-O-(coumaroyl)rutinoside-5-O-glucoside and Petunidin 3-O-(coumaroyl)rutinoside-5-O-glucoside) are also present in commonly consumed foods. Based on this, the anthocyanin content of Purple Tomato is not expected to present nutritional safety concerns.

Therefore, Health Canada has not identified any nutritional concerns related to the proposed food use of Purple Tomato.

Chemistry

The tomatine content of Purple Tomato was included as part of the compositional comparative assessment and was considered as part of the review conducted by Health Canada. Otherwise, chemical contaminant residue data have not been provided, nor have any unique contaminant considerations been identified with respect to Purple Tomato. As well, there are no maximum levels of relevance 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.

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, Health Canada 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 novel Del and Ros1 proteins have a regulatory role in the expression of anthocyanins in the Purple Tomato and do not have an enzymatic or structural role in the tomato. Their function in the tomato as transcription factors is to trigger the tomato's own endogenous tomato anthocyanin biosynthetic pathway, resulting in an increase in the production of the anthocyanins present in the tomato fruit.

A protein analysis using an Orbitrap LC-MS/MS was conducted on the Purple Tomato (ripe fruit). More than 2,700 peptides were detected but none corresponded to the Del or Ros1 proteins. These novel proteins were determined to be below the limits of detection of 4 ng/gfw for the Del protein and 1.5 ng/gfw for the Ros1 protein.

An in vitro digestion study was conducted using the Del and Ros1 proteins, using a simulated gastric fluid (SGF) assay with pepsin. The novel proteins from the Purple Tomato could not be isolated because they were present at levels too low to detect, so instead the transcription factors expressedin E. coli (strain BL21) were used. The results suggest that both the Del and Ros1 proteins are rapidly digested (<1 min and <10 min, respectively) in SGF.

Bioinformatics search for toxins was conducted on the amino acid sequences of the Del and Ros1 proteins using the UniProtKB/Swiss-Prot database. The BLASTP algorithm was used with the PAM30 matrix and an E-value threshold of 1x10-2. The parameters selected were considered to be sensitive and did not result in any matches of significant similarity to sequences of known toxins relevant to human health.

An additional bioinformatics search for toxins was conducted, using the Del and Ros1 protein sequences run against the ToxinPred 3.0 database of known toxic and non-toxic proteins, using a similarity-based BLAST search method and an E-value of 1x10-1. Again there were for no hits identified for toxins.

The level of chlorogenic acids (CGA) in the Purple Tomato ripe fruit compared to a wild-type tomato was determined to be about 2.5-fold higher (about 4.6 mg/100 gfw compared to 1.84 mg/100 gfw, respectively ^{Footnote 2}). However, the level of CGA in the Purple Tomato is still much lower than the levels found in other commonly consumed foods (e.g., raw blueberries have a mean range of 86.99 to 131.18 mg/100 gfw).

Alpha-tomatine is a naturally produced glycoalkaloid found in tomatoes and is a toxin which protects the tomato plant from attack by insects, fungi, etc. The level of alpha-tomatine in the Purple Tomato (i.e., a means of about 88 mg/kg dry weight [dw]) is within the OECD ^{Footnote 3} range for other varieties of tomato fruits (i.e., range of 0 to 389 mg/kgdw). Therefore, the level of alpha-tomatine in Purple Tomatoes is consistent with what would be consumed from other tomato varieties already on the market.

Based on the available data provided, Health Canada has no safety concerns regarding the Purple Tomato from a toxicological perspective.

Allergenicity

No priority allergens are utilized in the manufacturing of the Purple Tomato.

The petitioner demonstrated that the Ros1 and Del protein sequences exhibited no homology (>35%) to known allergens at the entire protein level.

Based on the available data provided, Health Canada has no safety concerns regarding the Purple Tomato from an allergenicity perspective.

Conclusion

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

Health Canada's opinion refers only to the food use of Purple Tomato. Issues related to its environmental release 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
Email: bmh-bdm@hc-sc.gc.ca