Agency Response Letter GRAS Notice No. GRN 000631
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See also Generally Recognized as Safe (GRAS).
CFSAN/Office of Food Additive Safety
July 18, 2016
Ms. Candice Cryne
AB Enzymes GmbH
Regulatory Affairs Specialist (The Americas)
Ontario, CANADA M6J3L9
Re: GRAS Notice No. GRN 000631
Dear Ms. Candice Cryne:
The Food and Drug Administration (FDA) is responding to the notice, dated February 1, 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). FDA received the notice on February 12, 2016, filed it on March 10, 2016, and designated it as GRAS Notice No. GRN 000631.
The subject of the notice is triacylglycerol lipase enzyme preparation produced by a genetically modified strain of Trichoderma reesei (triacylglycerol lipase enzyme preparation). The notice informs FDA of the view of AB Enzymes that triacylglycerol lipase enzyme preparation is GRAS, through scientific procedures, for use as an enzyme in the manufacture of baked goods, pasta, noodles, and cereal-based snack foods, at a maximum use level of 10 milligrams Total Organic Solids per kilogram (mg TOS/kg) of raw material.
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. AB Enzymes’ notice provides information about each of these components in the triacylglycerol lipase enzyme preparation.
According to the classification system of enzymes established by the International Union of Biochemistry and Molecular Biology, triacylglycerol lipase is identified by the Enzyme Commission Number 184.108.40.206. The accepted name for the enzyme is triacylglycerol lipase; and the systematic name is triacylglycerol hydrolase. This enzyme is also known as lipase; triglyceride lipase; tributyrase; butyrinase; glycerol ester hydrolase; tributyrinase; Tween hydrolase; steapsin; triacetinase; tributyrin esterase; Tweenase; amno N-AP; Takedo 1969-4-9; Meito MY 30; Tweenesterase; GA 56; capalase L; triglyceride hydrolase; triolein hydrolase; tween-hydrolyzing esterase; amano CE; cacordase; triglyceridase; triacylglycerol ester hydrolase; amano P; amano AP; PPL; glycerol-ester hydrolase; GEH; meito Sangyo OF lipase; hepatic lipase; lipazin; post-heparin plasma protamine-resistant lipase; salt-resistant post-heparin lipase; heparin releasable hepatic lipase; amano CES; amano B; tributyrase; triglyceride lipase; liver lipase; hepatic monoacylglycerol acyltransferase. The CAS Registry Number for triacylglycerol lipase is 9001-62-1. Triacylglycerol lipase catalyzes the hydrolysis of triacylglycerol to form diacylglycerol and free carboxylate.
AB Enzymes states that the triacylglycerol lipase gene from Fusarium oxysporum (Genbank CAB9359) was synthesized (Eurofins, Germany) using the T. reesei compatible Hypocrea jecorina optimized codon set. The recipient strain used in the construction of the production strain, T. reesei RF10625, is a genetically modified derivative of T. reesei RF4847 with a high capacity for triacylglycerol lipase production. Trichoderma reesei RF4847 has been taxonomically identified by the Dutch Culture Collection, CBS; an independent and internationally recognized laboratory. Trichoderma reesei RF4847 is constructed by transforming T. reesei RF7720 with a purified DNA fragment from a plasmid carrying the synthesized triacylglycerol lipase gene. AB Enzymes states that T. reesei is a nonpathogenic, nontoxigenic microbe, and that it has a long history of safe use for the production of enzymes used in food. AB Enzymes also states that the transformed DNA is stably integrated, and does not contain any antibiotic resistance genes. AB confirms that T. reesei RF10625 is genetically stable after ten generations.
AB Enzymes states that the triacylglycerol lipase enzyme preparation is produced by a controlled fed-batch submerged fermentation of a selected pure culture of the production strain. The manufacture of triacylglycerol lipase enzyme preparation includes fermentation, processing, and formulation of the final product. Appropriate measures are set in place to control identity, purity, and enzyme-generating ability of the production strain during and after fermentation. During fermentation the enzyme is secreted into the medium, and is separated by centrifugation or filtration, concentrated, and filtered at defined pH and temperature ranges. The enzyme concentrate is formulated as a liquid preparation with sunflower oil and wheat flour. According to AB Enzymes, the raw materials used in the fermentation, recovery, and formulation processes are food grade. The entire process is performed in accordance with Good Manufacturing Practice. AB Enzymes also states that the final triacylglycerol lipase enzyme preparation contains no major food allergens from the fermentation medium.
AB Enzymes states that the triacylglycerol lipase enzyme preparation is tested to ensure compliance with established specifications prior to release, including tests for the absence of the production organism in the final enzyme product. AB Enzymes notes that the triacylglycerol lipase enzyme preparation conforms to the specifications established for enzyme preparations in the Food Chemicals Codex (FCC, 9th edition, 2014; or FCC, 10th edition, 2016), 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 (JECFA, 2006). AB Enzymes provides data from two non-consecutive triacylglycerol lipase enzyme concentrate batches, to demonstrate that the manufacturing process conforms to set specifications.
AB Enzymes proposes to use triacylglycerol lipase enzyme preparation for baking applications including breads, biscuits, tortillas, cakes, steamed bread and croissants, and cereal-based processing, including pastas, noodles and snacks, at 10 mg TOS/kg of raw material. AB Enzymes states that no enzyme activity is present in the final food. However, in order to estimate dietary exposure to triacylglycerol lipase enzyme preparation, AB Enzymes assumes that the enzyme TOS will remain in the final food. Based on this assumption, AB Enzymes estimates the maximum daily intake of triacylglycerol lipase enzyme TOS from all intended food applications to be 0.09 mg TOS/kg body weight per day (mg TOS/kg bw/d) for a 60 kg adult. AB Enzymes states that triacylglycerol lipase enzyme activity will not produce reaction products that are not already part of the human diet.
AB Enzymes summarizes corroborative toxicological studies, using the triacylglycerol lipase enzyme concentrate, to support the safety of the triacylglycerol lipase enzyme preparation. Tests conducted using bacterial cells showed that triacylglycerol lipase enzyme is not mutagenic. AB Enzymes also demonstrates that the enzyme is not clastogenic to cultured human lymphocytes under the conditions employed in the study. The results of a 90-day oral toxicity study conducted using rats showed that consumption of triacylglycerol lipase enzyme concentrate did not cause any treatment-related adverse effects at 1000 mg/kg bw/d, the highest dose tested, which corresponds to 1000 mg TOS/kg bw/d of the triacylglycerol lipase enzyme. Based on the highest dose tested in the 90-day study, and the estimated maximum daily intake from the proposed use levels of triacylglycerol lipase enzyme preparation, i. e., 1000 mg TOS/kg bw/d and 0.09 mg TOS/kg bw/d, respectively, AB Enzymes calculates the margin of safety to be approximately 11,000.
AB Enzymes discusses potential food allergenicity of triacylglycerol lipase enzyme. AB Enzymes conducted an amino acid sequence homology search for triacylglycerol lipase enzyme against known allergens using the publicly available Food Allergy Research and Resource Program (FARRP) database, and the Allergen Database for Food Safety (ADFS). Amino acid identity matches greater than 35% over 80 amino acids were not found; no matches of contiguous stretches of eight amino acids shared between the triacylglycerol lipase enzyme amino acid sequence and known allergens were found. Based on the results obtained from the alignments and homology search results, AB Enzymes concludes that the triacylglycerol lipase enzyme does not show significant homology to any known allergen, and that the risk of allergic responses from oral consumption of triacylglycerol lipase enzyme is low.
Based on the data and information summarized above, AB Enzymes concludes that triacylglycerol lipase enzyme preparation is GRAS for its intended use.
The Federal Food, Drug, and Cosmetic Act (FD&C Act) requires 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)). The FD&C Act 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. Triacylglycerol lipase enzyme preparation produced by a genetically modified strain of T. reesei may require labeling under the FD&C Act, because it may contain protein derived from wheat. Questions about the submission of petitions or notifications for exemptions from food allergen labeling requirements should be directed to the Division of Biotechnology and GRAS Notice Review in the Office of Food Additive Safety. However, questions about food labeling in general should be directed to the Office of Nutrition, Labeling, and Dietary Supplements.
Standards of Identity
In the notice, AB Enzymes states its intention to use triacylglycerol lipase enzyme preparation in 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)
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 AB Enzymes’s notice that triacylglycerol lipase enzyme preparation produced by a genetically modified strain of T. reesei is GRAS for the intended uses, FDA did not consider whether section 301(ll) or any of its exemptions apply to foods containing triacylglycerol lipase enzyme preparation. Accordingly, this response should not be construed to be a statement that foods that contain triacylglycerol lipase enzyme preparation, if introduced or delivered for introduction into interstate commerce, would not violate section 301(ll).
Based on the information provided by AB Enzymes, as well as other information available to FDA, the agency has no questions at this time regarding AB Enzymes’ conclusion that triacylglycerol lipase enzyme preparation produced a genetically modified strain of T. reesei 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 triacylglycerol lipase enzyme preparation produced a genetically modified strain of T. reesei. As always, it is the continuing responsibility of AB Enzymes 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 00 0631, 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
 RF10625 has been deposited at Centraalbureau voor Schimmelcultures (CBS) as CBS 134213.
 RF4847 is a classical mutant derived from the well-characterized strain, T. reesei QM6a and has been deposited as CBS 114041.