Agency Response Letter GRAS Notice No. GRN 000472
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CFSAN/Office of Food Additive Safety
December 10, 2013
Ms. Lori Gregg
Novozymes North America, Inc.
77 Perry Chapel Church Road
P.O. Box 576
Franklinton, NC 27525
Re: GRAS Notice No. GRN 000472
Dear Ms. Gregg:
The Food and Drug Administration (FDA) is responding to the notice, dated April 24, 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 May 8, 2013, filed it on July 23, 2013, and designated it as GRAS Notice No. GRN 000472.
The subject of the notice is xylanase enzyme preparation derived from Bacillus licheniformis carrying a gene encoding a modified xylanase from B. licheniformis (xylanase enzyme preparation). The notice informs FDA of the view of Novozymes North America, Inc. (Novozymes) that xylanase enzyme preparation is GRAS, through scientific procedures, for use as an enzyme in baking applications at a level of 5 milligrams (mg) of xylanase enzyme preparation per kilogram (kg) of flour. Novozymes intends to use xylanase enzyme preparation to modify arabinoxylans for improved dough handling and bread characteristics.
Commercial enzyme preparations that are used in food processing typically contain an enzyme component, which catalyzes the chemical reaction that is 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 xylanase enzyme preparation.
According to the classification system of enzymes established by the International Union of Biochemistry and Molecular Biology, xylanase is identified by the Enzyme Commission Number 188.8.131.52. The accepted name for the enzyme is xylanase and the systematic name is endo-1, 4-beta-xylanase. The CAS Registry Number for xylanase is 9025-57-4. Xylanase catalyzes the hydrolysis of the xylosidic linkages in the arabinoxylan backbone resulting in depolymerization of arabinoxylans into smaller oligosaccharides.
Novozymes states that B. licheniformis strain BW302 is the host strain used to develop the production strain for xylanase enzyme. The host strain is derived from B. licheniformis strain ATCC 9789 using a targeted recombinant DNA technique to inactivate genes encoding several proteases and peptides, as well as genes essential for sporulation. Novozymes states that these modifications improve the safety and stability of xylanase enzyme. Novozymes also cites published studies describing B. licheniformis as a nonpathogenic and nontoxigenic microbe with a long history of safe industrial use for the production of enzymes used in human food. In addition, Novozymes cites several GRAS notices describing the food uses of enzymes derived from B. licheniformis. FDA had no questions in response to these GRAS notices.
Novozymes describes the development of the production strain, B. licheniformis strain HyGe329. The expression plasmid pBW120 was transformed into Bacillus subtilis strain BW154. This B. subtilis strain was then used as a donor to transfer plasmid pBW120 to the host strain via conjugation. Novozymes cites published information characterizing plasmid pBW120. The plasmid contains an expression cassette that includes: 1) a chemically-synthesized gene encoding a B. licheniformis xylanase enzyme with a single amino acid residue difference compared to the wild type sequence. Novozymes states that the gene was synthesized based on sequence data from SWISSPROT:052730, a public database; 2) a fragment of a hybrid promoter comprised of sequences from B. licheniformis, Bacillus amyloliquefaciens, and Bacillus thuringiensis, and 3) a B. licheniformis terminator sequence. The production strain contains two copies of the expression cassette at two 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 functional antibiotic resistance genes.
Novozymes states that xylanase 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, xylanase enzyme is secreted to the fermentation broth. After fermentation, the enzyme is recovered using multiple filtration and concentration steps that also remove residues of the production strain. Water, glycerol, sodium benzoate, and potassium sorbate are added to preserve and stabilize the enzyme concentrate. The enzyme concentrate is then formulated with wheat flour and sodium chloride to achieve the desired xylanase enzyme activity. Novozymes states that xylanase enzyme preparation does not contain any major food allergens from the fermentation medium.1
Xylanase enzyme preparation is sold as a granulated product. Novozymes states that xylanase enzyme preparation is produced in accordance with current Good Manufacturing Practices and the materials used in fermentation, recovery, and formulation meet predefined quality standards and are food grade.
Novozymes states that xylanase enzyme preparation conforms to the specifications for enzyme preparations described in the Food Chemicals Codex (8th edition, 2012) 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 (2006). Novozymes provided analytical data from one batch of xylanase enzyme preparation to demonstrate compliance with these specifications.
Novozymes intends to use xylanase enzyme preparation in flour at a maximum level of 5 mg (equivalent to 4% Total Organic Solids (TOS)) of xylanase enzyme preparation per kg of flour.2 Novozymes states that xylanase enzyme is inactivated during baking; thus, no xylanase enzyme activity is expected to remain in the final foods. However, to estimate dietary exposure to xylanase enzyme preparation, Novozymes assumes that all baked products are produced using xylanase enzyme preparation and that all of the enzyme would remain in the final foods. Based on these assumptions and the proposed maximum use level, Novozymes estimates the maximum daily intake of xylanase enzyme preparation from all the intended food applications to be 0.023 mg TOS per day, which corresponds to 0.376 x 10-3 mg TOS per kg body weight per day (mg TOS/kg bw/d).
In addition to published information describing the safety of the production strain and the characterization of the plasmid used in its development, Novozymes cites published information describing the long history of use of xylanases in food. Furthermore, Novozymes cites published studies demonstrating that enzymes do not generally raise safety concerns.
Novozymes summarizes unpublished toxicological studies supporting the safety of xylanase enzyme concentrate in the absence of standardization and stabilization ingredients. Tests conducted using bacterial cells showed that xylanase enzyme concentrate is not mutagenic. Tests conducted using human lymphocytes showed that xylanase enzyme concentrate is not clastogenic. The results of a 90-day oral toxicity study conducted using rats showed that consumption of xylanase enzyme concentrate did not cause adverse effects at 1020 mg TOS/kg bw/d, the highest dose tested. Novozymes designated this level as the no observed adverse effect level (NOAEL). Based on this NOAEL and the highest intended consumption level (0.376 x 10-3 mg/kg bw/d) for xylanase enzyme preparation, Novozymes calculates a margin of safety of 2.7 x 106.
Novozymes discusses the potential food allergenicity of xylanase enzyme. Novozymes conducted an amino acid sequence homology search for xylanase enzyme against known allergens in the Food Allergy Research and Resource Program database as well as the World Health Organization and the 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 xylanase enzyme amino acid sequence and those of known allergens. Based on this information, Novozymes considers it unlikely that oral consumption of xylanase enzyme will result in allergic responses.
Based on the data and information summarized above, Novozymes concludes that xylanase enzyme preparation is GRAS for the intended uses.
The Food Allergen Labeling and Consumer Protection Act of 2004 (FALCPA) amends the Federal Food, Drug, and Cosmetic Act (FD&C Act) to require that the label of a food that is or contains an ingredient that bears or contains a “major food allergen” declare the presence of the allergen (section 403(w)). FALCPA defines a “major food allergen” as one of eight foods or food groups (i.e., milk, eggs, fish, Crustacean shellfish, tree nuts, peanuts, wheat, and soybeans) or a food ingredient that contains protein derived from one of those foods. Xylanase enzyme preparation may require labeling under FALCPA, because it will contain protein derived from wheat. Although issues associated with labeling food are the responsibility of the Office of Nutrition, Labeling, and Dietary Supplements, issues associated with FALCPA exemptions are the responsibility of the Food Allergen Coordinator in the Office of Food Additive Safety.
Section 301(ll) of the 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 xylanase 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 xylanase enzyme preparation. Accordingly, this response should not be construed to be a statement that foods that contain xylanase 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 the information available to FDA, the agency has no questions at this time regarding Novozymes’ conclusion that xylanase 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 xylanase 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 000472, 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
1Novozymes also cites publicly available information discussing the lack of a health concern posed by the used of major food allergens in fermentation media used to produce microbially-derived enzyme preparations.
2% TOS = 100 - A - W - D; where: A = % ash, W = % water, and D = % diluent and/or formulation ingredients.