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See also Generally Recognized as Safe (GRAS).
CFSAN/Office of Food Additive Safety
November 23, 2016
Novozymes North America, Inc.
77 Perry Chapel Church Road
P.O. Box 576
Franklinton, NC 27525
Re: GRAS Notice No. GRN 000651
Dear Ms. Oesterling:
The Food and Drug Administration (FDA, we) completed our evaluation of GRN 000651. We received the notice, dated May 2, 2016, 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) on May 13, 2016, and filed it on June 6, 2016. We received an amendment containing clarifying information on October 25, 2016.
FDA published the GRAS final rule on August 17, 2016 (81 FR 54960), with an effective date of October 17, 2016. As GRN 000651 was pending on the effective date of the GRAS final rule, we requested some additional information consistent with the format and requirements of the final rule. We received an amendment responding to this request on October 24, 2016.
The subject of the notice is phospholipase A1 preparation produced by Aspergillus niger carrying a phospholipase A1 gene from Talaromyces leycettanus (phospholipase A1 enzyme preparation). The notice informs FDA of the view of Novozymes North America, Inc. (Novozymes) that phospholipase A1 enzyme preparation is GRAS, through scientific procedures, for use as an enzyme in the de-gumming of vegetable oil at a maximum use level of 2.96 milligrams Total Organic Solids per kilogram (mg TOS/kg) oil.
Commercial enzyme preparations that are used in food processing typically contain an enzyme component that catalyzes the chemical reaction as well as substances used as stabilizers, preservatives, or diluents. Enzyme preparations may also contain components derived from the production organism and components derived from the manufacturing process, e.g., constituents of the fermentation media or the residues of processing aids. Novozymes’ notice provides information about each of these components in the phospholipase A1 enzyme preparation.
According to the classification system of enzymes established by the International Union of Biochemistry and Molecular Biology, phospholipase A1 is identified by the Enzyme Commission Number 18.104.22.168. The accepted name for the enzyme is phospholipase A1, and the systematic name is phosphatidylcholine 1- acylhydrolase. The CAS Registry Number for phospholipase A1 is 9043-29-2. Phospholipase A1 catalyzes the hydrolysis of the sn-1 ester bond of diacylphospholipids to form 2-acyl-1-lysophospholipid and free fatty acids.
Novozymes states that the A. niger production strain, designated as 279-C2948-1, was derived from recipient strain A. niger C2948. Novozymes describes A. niger as a non-pathogenic, non-toxigenic, well- characterized production organism with a history of safe use in the food industry. Novozymes also states that A. niger is considered suitable for Good Industrial Large Scale Practice worldwide.
Novozymes describes that the construction of the recipient strain was done by deleting several genes, including amylases and proteases, in addition to the fumonisin gene cluster and the oxaloacetate hydrolase gene. Novozymes used an expression plasmid containing the phospholipase A1 gene from T. leycettanus (plA1), a fragment of the A. niger promoter, a transcriptional terminator, and the selective marker amdS to introduce the phospholipase A1 gene into the recipient strain. The expression plasmid was integrated into four specific loci by targeted homologous recombination. Novozymes states that all extraneous DNA was excised from the genome, leaving only the expression cassette of the plA1 gene containing the promoter and the transcriptional terminator at each target locus. Novozymes confirmed the sequence of the inserted expression cassettes and the flanking regions at each of the integration loci in the production strain by PCR analysis and sequencing. Novozymes states that they confirmed the genetic stability of the introduced DNA sequences by Southern blot hybridization, and that the transformed DNA is stably integrated into the A. niger chromosome, is poorly mobilized for genetic transfer, and is mitotically stable. Novozymes also used Southern blot hybridization to confirm that the production strain does not contain antibiotic resistance genes.
Novozymes states that the phospholipase A1 enzyme preparation is manufactured by submerged fermentation of a pure culture prepared from a stock culture of the production strain. Novozymes states that fermentation is carried out under controlled conditions and the culture is periodically tested to ensure production strain identity, purity, and enzyme-generating ability. After fermentation, the enzyme is recovered from the culture broth by a primary separation step after pH adjustment and the addition of a flocculating agent. The supernatant containing the enzyme is concentrated by ultrafiltration or evaporation. The concentrated enzyme solution is then filtered again to ensure removal of the production organism. After a final concentration step, the liquid enzyme concentrate is standardized with sorbitol and glycerol, and preserved by the addition of potassium sorbate and sodium benzoate. Novozymes states that the entire process is performed in accordance with current good manufacturing practices using raw materials of food grade quality. Novozymes also states that the final enzyme preparation contains no major food allergens from the manufacturing steps.
Novozymes has established food grade specifications and notes that the phospholipase A1 enzyme preparation conforms to specifications established for enzyme preparations in the Food Chemicals Codex (FCC, 9th edition, 2014), and to the General Specifications and Considerations for Enzyme Preparations Used in Food Processing established by the FAO/WHO Joint Expert Committee on Food Additives (JECFA, 2006). Novozymes provides analytical data from three batches of phospholipase A1 enzyme concentrate to demonstrate consistency with the specifications.
Novozymes intends to use the aqueous phospholipase A1 enzyme preparation in degumming of vegetable oil. Novozymes states that the degumming step in oil processing removes phospholipids, increases storage stability of the oil, and facilitates downstream processing. Novozymes states that phospholipase A1 enzyme preparation will not migrate into the oil phase. In addition, any residual enzyme preparation will be removed in the oil refining step. However, based on the maximum intended use level and the assumption that all of the phospholipase A1 enzyme preparation will remain in the final food, Novozymes estimates the dietary exposure to phospholipase A1 enzyme preparation to be 0.003 mg TOS/kg bodyweight per day (mg TOS/kg bw/d).
Novozymes relies on published information for the safety of microbial enzyme preparations used in food processing, the safety of the production organism, and provides a toxicity assessment of the phospholipase A1 enzyme preparation based on a bioinformatics analysis. Novozymes reports < 20% sequence homology when comparing the sequence of phospholipase A1 enzyme against known toxin sequences on the publicly available UNIPROT database. Novozymes also summarizes corroborative unpublished toxicological studies of the phospholipase A1 enzyme concentrate. Novozymes concludes that the phospholipase A1 enzyme is neither cytotoxic nor mutagenic based on results from an in vitro cytotoxicity assay and a bacterial reverse mutation assay, respectively.
Novozymes discusses the potential food allergenicity of phospholipase A1 enzyme. Novozymes conducted an 80-amino acid sequence homology search for phospholipase A1 enzyme against known allergens stored in the FARRP allergen protein database, and found no sequence identity matches over 35% to known allergens. Novozymes did not find any matches of contiguous stretches of eight amino acids in the phospholipase A1 enzyme sequence that would be cross reactive with an allergenic protein. Novozymes further discusses the conclusions of several organizations and working groups about the low risk of allergenicity posed by enzymes due to their low use levels and the extensive processing of enzyme-containing foods during manufacturing. Based on the totality of the information available, Novozymes concludes that it is unlikely that oral consumption of phospholipase A1 enzyme will result in allergenic responses.
Based on the data and information summarized above, Novozymes concludes that phospholipase A1 enzyme preparation is GRAS for its intended use.
Section 301(ll) of the Federal Food, Drug, and Cosmetic Act (FD&C Act)
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 our evaluation of Novozymes’ notice concluding that phospholipase A1 enzyme preparation is GRAS under its intended conditions of use, we did not consider whether section 301(ll) or any of its exemptions apply to foods containing phospholipase A1 enzyme preparation. Accordingly, our response should not be construed to be a statement that foods containing phospholipase A1 enzyme preparation, if introduced or delivered for introduction into interstate commerce, would not violate section 301(ll).
Based on the information that Novozymes provided, as well as other information available to FDA, we have no questions at this time regarding Novozymes’s conclusion that phospholipase A1 enzyme preparation produced by A. niger carrying a phospholipase A1 gene from T. leycettanus is GRAS under its intended conditions of use. This letter is not an affirmation that phospholipase A1 enzyme preparation produced by A. niger carrying a phospholipase A1 gene from T. leycettanus is GRAS under 21 CFR170.35. Unless noted above, our review did not address other provisions of the FD&C Act. Food ingredient manufacturers and food producers are responsible for ensuring that marketed products are safe and compliant with all applicable legal and regulatory requirements.
In accordance with 21 CFR 170.275(b)(2), the text of this letter responding to GRN 000651 is accessible to the public at www.fda.gov/grasnoticeinventory.
Dennis M. Keefe, Ph.D.
Office of Food Additive Safety
Center for Food Safety and Applied Nutrition
A. niger C2948 is an isolate of A. niger strain B0-1.
 The UNIPROT database contains entries from SWISSPROT and TREMBL PIR-PSD databases.