Agency Response Letter GRAS Notice No. GRN 000476
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CFSAN/Office of Food Additive Safety
February 3, 2014
Ms. Lori Gregg
Novozymes North America, Inc.
77 Perry Chapel Church Road
P.O. Box 576
Franklinton, NC 27525
Dear Ms. Gregg:
The Food and Drug Administration (FDA) is responding to the notice, dated May 30, 2013, that you submitted in accordance with the agency’s proposed regulation, proposed 21 CFR 170.36 (62 FR 18938; April 17, 1997; Substances Generally Recognized as Safe (GRAS); the GRAS proposal). FDA received the notice on June 4, 2013, filed it on June 20, 2013, and designated it as GRAS Notice No. GRN 000476.
The subject of the notice is asparaginase enzyme preparation from genetically modified Bacillus subtilis (asparaginase enzyme preparation). The notice informs FDA of the view of Novozymes North America, Inc. (Novozymes) that asparaginase enzyme preparation is GRAS, through scientific procedures, for use as an enzyme to reduce acrylamide formation in bread, potato-based snacks, breakfast cereals, coffee, and chocolate products.1 Novozymes proposes asparaginase enzyme preparation will be used at a maximum level of 20 milligrams Total Organic Solids per kilogram (mg TOS/kg) of food.
Commercial enzyme preparations that are used in food processing typically contain an enzyme component which catalyzes the chemical reaction responsible for the technical effect, as well as substances used as stabilizers, preservatives, or diluents. Enzyme preparations may also contain constituents derived from the production organism and constituents derived from the manufacturing process, e.g., components of the fermentation media or the residues of processing aids. Novozymes’ notice provides information about each of these components of asparaginase enzyme preparation.
According to the classification system of enzymes established by the International Union of Biochemistry and Molecular Biology, asparaginase is identified by the Enzyme Commission number 126.96.36.199. The accepted name for the enzyme is asparaginase and the systematic name is L-asparagine amidohydrolase. The CAS Registry Number for asparaginase is 9015-68-3. Asparaginase catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia. Asparaginase also acts on glutamine (with a lower preference). It has no activity on other amino acids or asparagine residues.
Novozymes states that B. subtilis strain PP2982 is the host strain used to develop the production strain. The host strain is derived from B. subtilis strain A164 using a targeted recombinant DNA technique to inactivate genes encoding several proteases, a gene essential for sporulation, and a geneessential for surfactin production.2 Novozymes states that these modifications aim to improve the safety and stability of asparaginase enzyme. Novozymes cites published information describing B. subtilis strains as nonpathogenic and nontoxigenic microbes. Novozymes also cites published information stating that B. subtilis is ubiquitous in nature, is a common component of human food, and has a history of safe industrial use for the production of enzymes used in human food. In addition, Novozymes cites two regulations (21 CFR 184.1148 and 21 CFR 184.1150) and several GRAS notices (GRNs 20, 114, 205, 274 and 406) describing the food uses of enzymes derived from B.subtilis. FDA had no questions in response to these GRAS notices.
Novozymes describes the development of the production strain, B. subtilis strain MOL2940. The host strain was transformed with expression plasmid pMOL2930. Plasmid pMOL2930 contains an expression cassette that includes: 1) a chemically-synthesized gene encoding an asparaginase enzyme based on that expressed by the extremophilic bacterium Pyrococcus furiosus. Novozymes states that the gene was synthesized based on sequence data published by the National Center for Biotechnology Information. Novozymes cites published literature describing this gene; 2) a fragment of a hybrid promoter comprised of sequences from Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus thuringiensis, and 3) a B. licheniformis terminator sequence. The production strain contains three copies of the expression cassette at three specific loci. According to Novozymes, Southern hybridization analyses confirmed that the expression cassette is stably integrated into the chromosome of the production strain and that the production strain does not contain genes encoding antibiotic resistance.
Novozymes states that asparaginase enzyme is produced by submerged, fed-batch fermentation of a pure culture of the production strain. Each fermentation batch is initiated using a lyophilized stock culture and appropriate measures are taken to control for identity, purity, and enzyme-generating ability before use. During fermentation, asparaginase enzyme is secreted to the fermentation broth. After fermentation, asparaginase enzyme is recovered by filtration and concentration steps that also remove residues of the production strain. Sodium chloride is added to stabilize the enzyme concentrate, which is then formulated with sodium benzoate and potassium sorbate. Either dextrin or a combination of water and sorbitol is added to the enzyme concentrate to produce granulated or liquid commercial products with the desired asparaginase enzyme activity. Novozymes states that asparaginase enzyme preparation is produced in accordance with current good manufacturing practices (cGMP). According to Novozymes, the raw materials used in the fermentation, recovery, and formulation processes meet predefined quality standards and are food grade. Novozymes states that asparaginase enzyme preparation does not contain any major food allergens from the fermentation medium.3
Novozymes notes that asparaginase enzyme preparation conforms to the specifications for enzyme preparations described in the Food Chemicals Codex (8edition, 2012) and to the current General Specifications and Considerations for Enzyme Preparations Used in Food Processing established by the FAO/WHO Joint Expert Committee on Food Additives (2006). Novozymes provided analytical data from one batch of asparaginase enzyme preparation to demonstrate compliance with these specifications.
Novozymes proposes use of their asparaginase enzyme preparation to replace other asparaginase enzyme preparations currently used in foods. The intended uses of the asparaginase enzyme preparation are in bread, potato-based snacks or chips, breakfast cereals, coffee, and chocolate products, at a maximum level of 20 mg TOS/kg of processed food. Novozymes states that asparaginase enzyme is inactivated during baking and frying of these foods; thus, no asparaginase activity is expected to remain in the final foods. However, to estimate dietary exposure to asparaginase enzyme preparation, Novozymes assumes that the asparaginase enzyme TOS will remain in the final foods. Based on this assumption, Novozymes estimates the maximum daily intake of asparaginase enzyme TOS from all the intended food applications to be 0.27 mg TOS/kg body weight per day (mg TOS/kg bw/d). Novozymes states that asparaginase enzyme activity will not produce reaction products that are not already part of the human diet.
In addition to published information describing the safety of the production strain and characterizing the gene encoding the asparaginase enzyme, Novozymes cites published information describing the presence of asparaginase enzymes in the normal human diet. Furthermore, Novozymes cites published studies demonstrating that enzymes used in foods do not generally raise safety concerns.
Novozymes summarizes unpublished toxicological studies supporting the safety of asparaginase enzyme concentrate in the absence of standardization and stabilization ingredients. Tests conducted using bacterial cells, mouse cells, and cultured human lymphocytes showed that asparaginase enzyme concentrate is noncytotoxic and nonmutagenic. The results of a 90-day oral toxicity study conducted using rats showed that consumption of up to 1207 mg TOS/kg bw/d (the highest dose tested) asparaginase enzyme concentrate does not cause adverse effects. Novozymes designated this level as the no observed adverse effect level (NOAEL). Based on this NOAEL and the highest intended consumption level (27 mg TOS/kg bw/d) for asparaginase enzyme preparation, Novozymes calculates a margin of safety of greater than 4000.
Novozymes discusses the potential food allergenicity of asparaginase enzyme. Novozymes conducted an amino acid sequence homology search for asparaginase enzyme against known allergens in the Food Allergy Research and Resource Program database and those in the World Health Organization and International Union of Immunological Societies Allergen Nomenclature Subcommittee database. No amino acid identity matches greater than 35% over 80 amino acids were found, nor were there any contiguous stretches of eight amino acids shared between the asparaginase enzyme amino acid sequence and those of known allergens. Based on this information, Novozymes considers it unlikely that oral consumption of asparaginase enzyme will result in allergic responses.
Based on the data and information summarized above, Novozymes concludes that asparaginase enzyme preparation is GRAS for the intended uses.
Standards of Identity
In the notice, Novozymes states its intention to use asparaginase enzyme preparation in several food categories, including foods for which standards of identity exist, located in Title 21 of the Code of Federal Regulations. We note that an ingredient that is lawfully added to food products may be used in a standardized food only if it is permitted by the applicable standard of identity.
Section 301(ll) of the Federal Food, Drug, and Cosmetic Act (FD&C Act)
The Food and Drug Administration Amendments Act of 2007 which was signed into law on September 27, 2007, amends the FD&C Act to, among other things, add section 301(ll). Section
301(ll) of the FD&C Act prohibits the introduction or delivery for introduction into interstate commerce of any food that contains a drug approved under section 505 of the FD&C Act, a biological product licensed under section 351 of the Public Health Service Act, or a drug or a biological product for which substantial clinical investigations have been instituted and their existence made public, unless one of the exemptions in section 301(ll)(1)-(4) applies. In its review of Novozymes’ notice that asparaginase enzyme preparation is GRAS for the intended uses, FDA did not consider whether section 301(ll) or any of its exemptions apply to foods containing asparaginase enzyme preparation. Accordingly, this response should not be construed to be a statement that foods that contain asparaginase enzyme preparation, if introduced or delivered for introduction into interstate commerce, would not violate section 301(ll).
Based on the information provided by Novozymes, as well as other information available to FDA, the agency has no questions at this time regarding Novozymes’ conclusion that asparaginase enzyme preparation is GRAS under the intended conditions of use. The agency has not, however, made its own determination regarding the GRAS status of the subject use of asparaginase enzyme preparation. As always, it is the continuing responsibility of Novozymes to ensure that food ingredients that the firm markets are safe, and are otherwise in compliance with all applicable legal and regulatory requirements.
In accordance with proposed 21 CFR 170.36(f), a copy of the text of this letter responding to GRN 000476, as well as a copy of the information in this notice that conforms to the information in the GRAS exemption claim (proposed 21 CFR 170.36(c)(1)), is available for public review and copying at www.fda.gov/grasnoticeinventory.
Dennis M. Keefe, Ph.D.
Office of Food Additive Safety
Center for Food Safety and Applied Nutrition
1Acrylamide forms when foods containing asparagine and reducing sugars are baked or fried at temperatures above 120°C. Novozymes intends to use asparaginase enzyme preparation to reduce L-asparagine levels in the proposed foods, thereby reducing acrylamide levels in these foods. FDA has not evaluated the efficacy of asparaginase enzyme preparation.
2Novozymes states that surfactins belong to the lipopeptide family, and have been previously considered to be a safety concern for B. subtilis strains. According to Novozymes, even though subsequent toxicological evidence has not demonstrated risk associated with surfactin, an added reason for the removal of the gene encoding for surfactin production was aimed at reducing foaming during fermentation.
3Novozymes also cites publicly available information discussing the lack of a health concern posed by the use of major food allergens in fermentation media used to produce microbially-derived enzyme preparations.