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Decision Document DD2016-117
Determination of the Safety of AquaBounty Technologies Inc.'s Salmon (Salmo salar L.) Event

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May 19, 2016

This Decision Document has been prepared to explain the regulatory decisions reached under the Feeds Regulations.

The Animal Feed Division (AFD) of the Animal Health Directorate of the Canadian Food Inspection Agency (CFIA) has evaluated information submitted by AquaBounty Technologies Inc. This information concerns the AquAdvantage® salmon (AAS) event which displays enhanced growth compared to conventional salmon. The CFIA has determined that feed ingredients derived from this animal with a novel trait does not present livestock feed safety or nutrition concerns when compared to feeds derived from salmon currently permitted to be used as livestock feed in Canada.

Taking into account this evaluation, feed ingredients derived from AAS therefore authorized by the AFD of the Animal Health Directorate of the CFIA as of May 19, 2016 for use in livestock feeds. Livestock feed ingredients may be derived from any salmon lines originating from AAS, provided that (i) no inter-specific crosses are performed, (ii) the intended uses are similar, (iii) it is known, based on characterization, that these animal do not display any additional novel traits and feed ingredients derived from these animals (e.g., fish meal and fish oil) are substantially equivalent to those that are currently permitted to be used as livestock feed in Canada, in terms of their livestock feed safety and nutrition, (iv) the novel genes are expressed at a level similar to that of the authorized event, and (v) data used to establish the substantial equivalence of lines derived from AAS be made available to the CFIA upon request.

AAS is subject to the same zoosanitary import requirements as unmodified salmon. AAS is required to meet the requirements of other jurisdictions, including but not limited to, the Food and Drugs Act and the Canadian Environmental Protection Act.

Please note that the livestock feed assessment of novel feeds is a critical step in the potential commercialization of this genetically modified animal. Other requirements, such as the assessment of novel foods by Health Canada and the environmental safety assessment by Environment and Climate Change Canada and Fisheries and Oceans, have been addressed separately from this review.

This bulletin was created by the Canadian Food Inspection Agency. For further information, please contact the Animal Feed Division by visiting the contact page.

Table of Contents

  1. Brief Identification of the Modified Animal
  2. Background Information
  3. Description of the Novel Trait
  4. Criteria for the Livestock Feed Assessment
  5. New Information Requirements
  6. Regulatory Decision

I. Brief Identification of the Modified Animal

Designation of the Modified Animal:
AquAdvantage® salmon event
Applicant:
AquaBounty Technologies Inc.
Animal Species:
Salmon (Salmo salar L.)
Novel Trait:
Enhanced growth
Trait Introduction Method:
Micro-injection of salmon eggs
Intended Use of the Modified Animal:
AquAdvantage® salmon event is intended to be raised for traditional salmon human food and livestock feed uses.

II. Background Information

AquaBounty Technologies Inc. has developed the AquAdvantage® salmon (AAS) event which displays enhanced growth during early life, compared to conventional salmon; growth rate normalizes in older animals. This enhanced growth rate in early life reduces the time to market of the salmon. AAS was developed by AquaBounty Technologies Inc. using recombinant deoxyribonucleic acid (DNA) technology, resulting in the introduction into Atlantic salmon (Salmo salar) of a growth hormone 1 (GH-1) gene from Chinook salmon (Oncorhynchus tshawytscha) under control of regulatory sequences of an antifreeze protein (AFP) gene from the ocean pout (Macrozoarces americanus). The Chinook salmon GH-1 protein in AAS operates within a regulatory framework of less tissue specificity than native growth hormone expression in Atlantic salmon, resulting in the enhanced growth displayed by AAS in early life.

AquaBounty Technologies Inc. has indicated that they intend to market AAS in the form of all-female predominately triploid, eyed-eggs. These eggs would be generated from an all genetic female population by sex-reversal of genetic females through17α-methyltestosterone treatment to produce phenotypic males for breeding and through hydrostatic pressure shock of the resulting fertilized eggs to induce triploidy. Hydrostatic pressure shock is not 100% effective at inducing triploidy, thus it is expected that a small proportion of the eggs would be diploid. The eyed eggs would be used to produce commercial weight salmon in in-land tank aquaculture rearing systems in Panama by the purchasers of the eggs. Feed ingredients would be derived predominantly from adult AAS.

AquaBounty Technologies Inc. has provided information on the identity of AAS; a detailed description of the transformation method; and information on insert copy number and intactness, levels of protein expression in the animal and the role of the inserted gene and regulatory sequences. The novel protein was identified and characterized. Information was provided for the evaluation of the potential toxicity of the novel protein to livestock and non-target organisms and potential allergenicity of the novel protein to humans and to livestock.

Ten independent crosses of AAS were performed over the 1997, 1998, and 1999 breeding seasons. Muscle-skin samples were collected from AAS derived from these crosses and from unmodified control salmon collected from the same breeding facility and possessing the same genetic background as AAS. Samples were also collected from unmodified reference salmon collected from commercial salmon farms in Maine and New Brunswick, Canada for use as study comparators. Nutritional components of AAS, such as proximates, vitamins, minerals, amino acids, and fatty acids, and levels of growth hormones and other hormones in the salmon were compared to those of the unmodified control salmon and reference salmon.

The AFD has considered both intended and unintended effects and similarities and differences between AAS and unmodified salmon relative to the safety and nutrition of feed ingredients derived from AAS for their intended purpose, including:

The AFD has also considered whether feeds derived from AAS meet the definitions and requirements of feeds as listed in Schedule IV of the Feeds Regulations.

III. Description of the Novel Trait

1. Development Method

AAS was developed through micro-injection of a transgene containing the gh-1 gene from Chinook salmon and regulatory sequences of an AFP gene from ocean pout into the eggs of wild-type Atlantic salmon. AAS was identified as a successful transformant based on molecular analyses and phenotypic testing for enhanced growth and was subsequently developed into a commercial line through multi-generational crosses with domesticated Atlantic salmon broodstock common to Atlantic Canada.

All-female triploid, eyed-eggs are generated from an all genetic female population by sex-reversal of genetic females through17α-methyltestosterone treatment to produce phenotypic males for breeding. Triplody is used in the aquaculture industry as a means of genetic containment as triploid fish are sterile. Triploidy is induced in resulting fertilized eggs through hydrostatic pressure shock; although, this is not 100% effective at inducing triploidy, thus it is expected that a small proportion of the eggs would be diploid. Eyed eggs would be used to produce commercial weight salmon in in-land tank aquaculture rearing systems with feed ingredients being derived predominantly from adult AAS.

2. Enhanced Growth

Growth hormones promote increased growth in salmonids. Growth hormones induce this response either by acting directly on cells or by inducing production of another hormone known as insulin-like growth factor (IGF). In conventional salmon, growth hormone expression is associated only with the pituitary gland. In contrast, the ocean pout AFP gene, whose regulatory sequences control expression of Chinook salmon GH-1 protein in AAS, lacks tissue specificity. Thus, the GH-1 protein in AAS is present within a regulatory framework of less tissue specificity than native growth hormone expression in Atlantic salmon, resulting in enhanced growth in early life. AAS grow 5 to 10 times faster than unmodified salmon during their pre-smolt stage and have the potential to reach market size a year earlier.

The Chinook salmon GH-1 protein introduced into AAS shares 94.1% sequence identity with the GH-1 protein of Atlantic salmon. Feed ingredients such as fish meal and fish oil may be derived from Chinook salmon and Atlantic salmon (among other species) and are listed livestock feed ingredients in Schedule IV of the Feeds Regulations in Canada, thus there is a history of livestock feed exposure to GH-1 proteins of both species.

Overall growth hormone protein expression in muscle-skin samples of market-size AAS, unmodified control salmon, and farmed reference salmon was measured by immunoassay. None of the samples possessed growth hormone protein at levels above the lower limit of quantification of the assay (10.40 ng/mL). Tissue-specific gene expression of GH-1 protein in AAS was assessed by Northern blots and Reverse Transcriptase Polymerase Chain reaction (RT-PCR) assays. The results of highly sensitive RT-PCR assays indicated that growth hormone genes were expressed in multiple tissues. In contrast, in less sensitive Northern blots, growth hormone gene expression was detected only in spleen and pituitary gland tissues, and the pituitary gland tissue expression could be attributed to endogenous growth hormone gene expression. The results of these studies indicate that Chinook GH-1 expression in AAS is less tissue-specific than endogenous growth hormone expression but that growth hormone expression occurs at very low levels.

The potential allergenicity and toxicity of the GH-1 protein to livestock and non-target organisms were evaluated. The weight of evidence indicates that the GH-1 protein is unlikely to be allergenic, based on the following information. With the exception of the parvalbumin allergen, found in all salmon, the Chinook salmon source of the GH-1gene is not associated with allergenicity. The parvalbumin gene was not introduced into AAS. In addition, parvalbumin levels in AAS muscle-skin samples were quantified and although moderately increased in these samples, their levels fell within the variation typically observed for similar quantification methods and did not exceed levels observed in other fish products. The amino acid sequence of the GH-1 protein itself was found to lack relevant similarities to known allergens. It was also concluded that the GH-1 protein is unlikely to be toxic to livestock and non-target organisms because it lacks a mode of action to suggest that it is intrinsically toxic to livestock or non-target organisms. For a more detailed discussion of the potential allergenicity and toxicity of the GH-1 protein, see Section IV, part 2.

3. Stable Integration into the Animal Genome

Molecular characterization by Southern blot analysis and DNA sequencing demonstrated that AAS contains one DNA insert containing the GH-1 gene and the AFP gene regulatory elements inserted at a single site in the salmon genome. A second non-functional DNA insert was detected in progeny of the initial AAS but was bred out of the line in subsequent generations. A 5' to 3' rearrangement of the initial portion of the 5' untranslated region of the AFP gene regulatory elements of the functional insert was found to have occurred during genomic integration into AAS. No truncation or frame-shift mutation of the native coding domain or generation of newly expressed fusion proteins was anticipated as a result of this rearrangement. No backbone sequences from the plasmid vector, linked or unlinked to the intact insert, were detected in AAS.

The stability of the insert within AAS over three generations was verified by multiplex Polymerase Chain Reaction (mPCR) assays and DNA sequencing, and the stability of the rapid growth phenotype was also confirmed in these generations. The inheritance pattern of the rapid growth phenotype indicated that the active DNA insert in AAS segregates according to Mendelian rules of inheritance for a single genetic locus.

IV. Criteria for the Livestock Feed Assessment

The AFD considered nutrient and anti-nutrient profiles; the safety of feed ingredients derived from AAS, including the presence of gene products, residues and metabolites in terms of animal health and human safety as it relates to the potential transfer of residues into foods of animal origin and worker/bystander exposure to the feed; and whether feeds derived from AAS meet the definitions and requirements of feeds as listed in Schedule IV of the Feeds Regulations. The potential impact of feeds derived from AAS on the environment was also assessed.

1. Potential Impact of AquAdvantage® Salmon Event on Livestock Nutrition

Nutrient and anti-nutrient composition:

The nutritional equivalence of AAS (TX) was compared to non-transgenic Farm Control (FC) and Sponsor Control (SC) salmon in a study conducted in Canada from October 2001 to October 2004. The study was a single-blind, comparator-controlled, quantitative determination of the nutritional composition of edible tissues (muscle-skin) in market-size diploid and triploid, male and female AAS. Edible tissue samples were collected for compositional analyses. Collected samples were analysed for proximates (protein, total fat, carbohydrate, moisture, and ash), vitamins, minerals, fatty acids, and amino acids. Compositional data were analysed statistically using analysis of variance (ANOVA) and statistically significant differences between the TX and SC were identified (P < 0.05) using the complete data set and subsets specific to the description of AAS, specifically diploid and triploid females. Where statistically significant differences were identified, Ranges of TX values were also assessed within the context of the natural variability of conventional salmon composition compiled from nutrient tables that included:

Additionally, an analysis of these data, conducted by the United States Food and Drug Administration, Center for Veterinary Medicine (CVM) Briefing Packet on AquAdvantage Salmon for the Veterinary Medicine Advisory Committee (September 20, 2010) on the Summary of the Compositional Analysis Results of Study AA-HFS-001 was also used during the review.

Overall, comparison of the AAS complete compositional data set (i.e., FC, SC and TX market-size diploid and triploid, male, and female) with the compiled nutrient tables showed that both for minimum and maximum values for all analytes were within the range for conventional salmon composition. Only niacin values fell outside the compositional range identified in the compiled nutrient tables. However, the potential increases in niacin consumption resulting from intake of feeds derived from AAS would not pose a nutritional concern.

Analysis of the subset of compositional data comparing triploid female TX to the SC group identified the following:

While statistically significant differences compared to controls were identified, these differences were not compositionally meaningful as they were within the ranges of values for conventional salmon composition range in the compiled nutrient tables.

Analysis of the subset of compositional data comparing diploid female TX to the SC group identified the following:

While statistically significant differences compared to controls were identified, these differences were not compositionally meaningful as they were within the ranges of values for conventional salmon composition range in the compiled nutrient tables.

Conclusion

It was concluded, based on the evidence provided by AquaBounty Technologies Inc. that the nutritional composition of the AAS is similar to that of the sponsored control and farm control salmon and to salmon in the commercial market place. Furthermore, it is concluded that feed ingredients derived from AAS will have nutritional composition equivalent to that of those feed ingredients derived from conventional salmon.

2. Potential Impact of AquAdvantage® Salmon Event on Animal Health and Human Safety as it Relates to the Potential Transfer of Residues into Foods of Animal Origin and Worker/Bystander Exposure to the Feed

AAS displays enhanced growth during early life due to altered expression of the GH-1 protein. The assessment of AAS evaluated the impact of the following potential hazards relative to the safety of feed ingredients derived from this event:

Chinook salmon GH-1 protein:

The potential allergenicity and toxicity of the Chinook salmon GH-1 protein to livestock and non-target organisms were evaluated. The weight of evidence indicates that this GH-1 protein is unlikely to be allergenic, based on the following information. With the exception of the parvalbumin allergen, found in all salmon, the Chinook salmon source of the GH-1 gene is not associated with allergenicity. The parvalbumin gene was not introduced into AAS. In addition, although quantification of parvalbumin in AAS muscle-skin samples indicated that parvalbumin levels were moderately increased in these samples, these increased levels fell within the variation typically observed for similar quantification methods and did not exceed levels observed in other fish products. The amino acid sequence of the GH-1 protein itself was found to lack relevant similarities to known allergens.

It was also concluded that this GH-1 protein is unlikely to be toxic to livestock and non-target organisms because it lacks a mode of action to suggest that it is intrinsically toxic to livestock or non-target organisms and considering that incidental exposure to Chinook and Atlantic salmon GH-1 proteins present in muscle-skin has occurred among livestock consumers of feeds derived from these fish. Also, evidence was provided that peptide hormones have poor bioavailability when consumed orally as they are hydrolyzed in the gut, that growth hormones of lower vertebrates do not bind the growth hormone receptor of higher vertebrates with sufficient affinity to activate the signalling cascade, and that peptide hormones would be heat labile and thus denatured during processing into fish meal, the form of feed that would most likely contain proteins derived from AAS in Canada. This information suggested that the exposure and bioavailability of Chinook salmon GH-1 protein in livestock would be reduced, which was also supportive of safety of feeds derived from AAS.

In addition to these factors, assessment of the potential toxicity of the Chinook salmon GH-1 protein also considered the health status of AAS. Animal health is an indicator of the safety of derived feeds and the practice of only allowing animals with known and acceptable health status to enter the feed supply is an essential step to ensuring safe feed. It was concluded in an animal health assessment performed by Health Canada that AAS do not have a diminished capacity to survive at any life stage nor are less resistant to microbial infection. The submitted data showed that both GE and non-GE fish had slight to moderate morphological irregularities. These types of irregularities observed do occur in farm-raised Atlantic salmon. Even though there was a higher incidence of these irregularities in the GE fish, Health Canada's assessment concluded that these fish are as likely to survive to market weight and be no more susceptible to disease. Thus the safety of feed products derived from AAS is supported by health status assessment.

Livestock exposure to the Chinook GH-1 protein is expected to be negligible as growth hormones are expressed at very low levels in AAS and would be further degraded during heat processing and passage through the gut, as indicated above.

Impact of novel GH-1 protein on IGF and other hormones:

Growth hormones promote increased growth either by acting directly on cells or by inducing production of IGF. Levels of IGF, and other hormones that affect growth and metabolism (estradiol, testosterone, 11-keto-testosterone, triiodothyronine, and trtraiodothyronine) were also measured by immunoassays in the same AAS, SC, and FC, salmon tissues that were tested for growth hormone levels. None of these hormones were expressed at significantly different levels in AAS than in the SC or FC reference salmon. This suggests that the increased expression of GH-1 in AAS did not have a significant impact on these other hormones levels.

In addition, the same evidence that peptide hormones have poor bioavailability when consumed orally as they are hydrolyzed in the gut and that peptide hormones would be heat labile and thus denatured during processing into fish meal, and the lack of adverse effects observed during the animal health assessment of AAS all suggest that increased levels of GH-1 did not affect IGF or other hormones in a manner that would impact the safety of feeds derived from AAS.

Altered Disease and Contaminant Profile in Feeds Derived from AAS

The Veterinary Drugs Directorate of Health Canada reviewed the AAS submission as it relates to animal health. The data provided demonstrated the AAS were as healthy as non-transgenic salmon. Data submitted was of considerable volume and collected over 11 years and provide no indication of diminished capacity of AAS to survive at any life-stage or be more susceptible to microbial infections. Therefore, AAS would not be expected to require more veterinary drugs compared to conventional aquaculture salmon. As such, feeds derived from AAS are no more likely to serve as a disease vector than those ingredients derived from wild or conventional farmed salmon nor have a significantly altered drug residue profile than those derived from conventional farmed salmon.

Exposure to environmental contaminants such as pesticides, heavy metals, and organic pollutants in farmed and wild salmon is primarily a function of dietary exposure, sources, and chemical form. Feeds produced for AAS are derived from the same ingredients used in conventional aquaculture and intake over the production lifetime is comparable. Environmental contaminants are not expected to be taken up, accumulate, distribute or sequester in tissues in a manner different from other farmed salmon and in the case of heavy metals and persistent organic pollutants are more likely to accumulate to a lesser degree as compared to wild Atlantic salmon due to shorter lifetime. Compositional analysis of AAS was found the be similar to that of the farm control and sponsor control salmon and to salmon in the commercial market place (see Section IV, Part I) and as such, feed ingredients derived from AAS are not expected to have significantly different contaminant profile from those ingredients derived from wild or conventional farmed salmon.

3a. Potential impact of Feeds derived from AquAdvantage® Salmon Event on the Environment

Feed ingredients such as fish meal and fish oil derived from AAS have been assessed to be equivalent to those derived from conventional fish sources in terms of their nutritional composition and contaminant profiles. Therefore, the use of these ingredients in livestock feeds is comparable to conventional feed ingredients derived from fish with no greater impact on the environment.

3b. Potential impact of AquAdvantage® Salmon Event on the Environment

The Canadian Environmental Protection Act, 1999 (CEPA 1999), administered by Environment and Climate Change Canada (ECCC) and Health Canada (HC), is the key authority for the Government of Canada to ensure that all new substances, including organisms, are assessed for their potential harm to the environment and human health. The New Substances Notification Regulations (Organisms) [NSNR (Organisms)] under CEPA 1999 prescribe the information that must be provided to ECCC prior to the import or manufacture in Canada of new organisms that are animate products of biotechnology, including fish products of biotechnology.

Fisheries and Oceans Canada (DFO), ECCC and HC have a Memorandum of Understanding respecting the implementation of the NSNR (Organisms) for fish. DFO assists in implementing the NSNR (Organisms) by conducting an environmental and indirect human health risk assessment for fish products of biotechnology and recommending any necessary measures to manage risks. The risk assessment conducted by DFO was peer-reviewed by an independent panel of experts under the auspices of the Canadian Science Advisory Secretariat.

The DFO completed their environmental risk assessment and determined that AAS was not "CEPA toxic"; in other words that there was no concern for the environment or indirect human health from the contained production of these fish. This decision allows AquaBounty Technologies Inc. to produce sterile eyed-eggs in Canada in contained facilities. Any other growth of the fish outside of the approved contained production conditions would require another notification and is not currently allowed under the NSNR (Organisms).

Summary of the environmental and indirect human health risk assessment of AquAdvantage® salmon PDF (264 kb)

Conclusion:

It was concluded, based on the evidence provided by AquaBounty Technologies Inc., that the novel GH-1-based enhanced growth trait will not confer to AAS any characteristic that would raise concerns regarding the safety of feed derived from AAS. Feed ingredients derived from AAS are considered to meet present ingredient definitions such as fish meal and fish oil derived from conventional salmon.

V. New Information Requirements

If at any time, AquaBounty Technologies Inc. becomes aware of any new information regarding risk to the environment, livestock or human health, which could result from the unconfined environmental release or livestock feed use of AAS or lines derived from it, AquaBounty Technologies Inc. is required to immediately provide such information to the CFIA. On the basis of such new information, the CFIA will re-evaluate the potential impact of AAS on the environment, livestock and human health and may re-evaluate its decision with respect to the livestock feed use and unconfined environmental release authorizations of AAS.

VI. Regulatory Decision

Based on the review of the data and information submitted by AquaBounty Technologies Inc. and input from other relevant scientific sources, the AFD of the Animal Health Directorate, CFIA, has concluded that the novel Chinook GH-1 protein-based enhanced growth trait will not confer to AAS any characteristic that would raise concerns regarding the safety or nutrition efficacy of AAS. Livestock feeds derived from salmon are currently listed in IV of the Feeds Regulations. AAS has been found to be as safe as and as nutritious as currently and historically grown salmon varieties. AAS and its products are considered to meet present ingredient definitions.

Taking into account this evaluation, feed ingredients derived from AAS therefore authorized by the AFD of the Animal Health Directorate of the CFIA as of May 19, 2016 for use in livestock feeds. Livestock feed ingredients may be derived from any salmon lines originating from AAS , provided that (i) no inter-specific crosses are performed, (ii) the intended uses are similar, (iii) it is known, based on characterization, that these animal do not display any additional novel traits and feed ingredients derived from these animals (e.g., fish meal and fish oil) are substantially equivalent to those that are currently permitted to be used as livestock feed in Canada, in terms of their livestock feed safety and nutrition, (iv) the novel genes are expressed at a level similar to that of the authorized event, and (v) data used to establish the substantial equivalence of lines derived from AAS be made available to the CFIA upon request.

AAS is subject to the same zoosanitary import requirements as unmodified salmon. AAS is required to meet the requirements of other jurisdictions, including but not limited to, the Food and Drugs Act and the Canadian Environmental Protection Act.

Please refer to Health Canada's Decisions on Novel Foods for a description of the food safety assessment of AAS.

Summary of the environmental and indirect human health risk assessment of AquAdvantage® salmon PDF (264 kb)

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