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CFSAN/Office of Food Additive Safety
October 1, 2009
1601 K Street NW
Washington, DC 20006-1600
Re: GRAS Notice No. GRN 000296
Dear Mr. Yingling:
The Food and Drug Administration (FDA) is responding to the notice, dated July 08, 2009, 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 July 10, 2009, filed it on July 10, 2009, and designated it as GRAS Notice No. GRN 000296.
The subject of the notice is a lipase enzyme preparation derived from a genetically modified strain of Aspergillus niger. The notice informs FDA of the view of DSM Food Specialties (DSM) that the lipase enzyme preparation is GRAS, through scientific procedures, for use in baked goods to enhance the gas holding capacity of the dough, leading to an increased stability of the dough upon proofing.
Commercial enzyme preparations that are used in food typically contain an enzyme component, which catalyzes the chemical reaction that is responsible for its technical effect, as well as substances used as stabilizers, preservatives or diluents. Enzyme preparations may also contain constituents derived from the production organism and manufacturing process. In its notice, DSM provides information about all the components of the lipase enzyme preparation.
The principal activity of DSM’s lipase is the hydrolysis of a triacylglycerol to a diacylglycerol and a carboxylate. The enzyme can also catalyze the hydrolysis of other lipid substrates including diacyl lipids, phospholipids and glycolipids. According to the classification system of enzymes established by the International Union of Biochemistry and Molecular Biology (IUBMB), lipase is identified by the Enzyme Commission number 220.127.116.11. Its accepted name is triacylglycerol lipase, and its systematic name is triacylglycerol acylhydrolase. The Chemical Abstract Service Registry number for lipase is 9001-62-1. DSM notes that the lipase produced by genetically modified A. niger is a glycoprotein with a processed sequence of 274 amino acids, and a calculated molecular mass of 28.7 kilodaltons (kDa).
DSM states that the production organism, A. niger, is nonpathogenic and nontoxigenic and has a long history of use as a source of enzymes used in food. DSM describes the development of the lipase production strain designated as LFS-54 from the host strain, ISO-528.
DSM describes the derivation of the host strain ISO-528. The A. niger strain NRRL 3122 is initially modified by classical mutagenesis to obtain strain GAM-53. DSM states that the GAM-53 strain is fully characterized and has been used to generate other ISO- strains that, in turn, were used to construct the production strains for enzymes such as phospholipase A2 (GRN 000183), and asparaginase (GRN 000214). The GAM-53 strain is further modified using molecular biology techniques to create “plug sites” for gene insertion, inactivate major amylase and protease genes, and improve the enzyme secretion capacity.
DSM states that the host strain ISO-528 was transformed with two expression cassettes. One of the cassettes contained the synthetic pre-pro-lipase gene based on the pre-pro-lipase genes of various Fusarium species. The other cassette contained the selectable marker gene amdS from Aspergillus nidulans. The amdS gene was subsequently removed to create a marker-free production strain. DSM states that the final production strain, LFS-54, contains multiple copies of the pre-pro-lipase gene and is genetically stable. DSM also states that specific tests have been performed to confirm that the LFS-54 strain does not produce any known toxins under fermentation conditions used in the production of lipase as well as under conditions known to induce toxin synthesis.
DSM states that lipase is produced by pure culture fermentation of the production strain LFS-54. The fermentation process consists of three steps: pre-culture fermentation, seed fermentation, and main fermentation. The fermentation is conducted under controlled conditions and is periodically tested for microbial contamination. Lipase is secreted to the fermentation broth and is subsequently purified, concentrated, and spray-dried. The dry product is formulated with wheat flour and standardized to the desired lipase activity. The content of total organic solids (TOS) of the final enzyme preparation is approximately 4.5 percent. DSM states that the manufacturing process is performed accordance with Good Manufacturing Practice. The raw materials used in the fermentation and recovery meet predefined quality standards and the raw materials used for the formulation are food grade.
DSM states that that the lipase enzyme preparation conforms to the specifications for enzyme preparations described in the Food Chemicals Codex (6th edition) and to the current (2006) General Specifications and Considerations for Enzyme Preparations Used in Food Processing established by the FAO/WHO Joint Expert Committee on Food Additives. The lipase enzyme preparation does not contain antibiotics and transformable recombinant DNA.
DSM states that the lipase enzyme preparation will be used in bakery products, where lipase will catalyze the conversion of apolar triglycerides present in flour or added to the flour to more polar di- and monoglycerides resulting in increased crumb softness and regularity. Lipase can also convert less polar, less emulsifying diacyl phospho- and galactolipids present in the flour to their highly polar, strongly emulsifying monoacyl counterparts. The lipase enzyme preparation will be used at levels of 14-164 DLU per kilogram flour, where DLU is a lipase activity unit. These use levels correspond to 0.14-2.37 milligrams TOS per kilogram finished food. DSM estimates the maximum daily intake of the lipase to be 0.00-0.01 milligram TOS per kilogram body weight per day (mg TOS/kg bw/d) based on the assumption that 100 percent of the enzyme TOS added to flour would remain in baked goods. Lipase is expected to be inactivated during baking. DSM does not anticipate that the action of lipase will result in the formation of any reaction products that are not already part of the human diet.
DSM summarizes unpublished toxicological studies conducted with the non-formulated enzyme concentrate. The studies included 14-day and 90-day oral toxicity tests in rats, a bacterial reverse mutation assay, and two in vitro mammalian chromosomal aberration tests. DSM states that the enzyme concentrate was not toxic in both 14-day and 90-day studies and not mutagenic in the reverse mutation assay. The results of the chromosomal aberration tests show that the enzyme concentrate is not clastogenic to cultured human lymphocytes.
DSM discusses potential allergenicity of enzymes used in food. DSM states that the levels of residual enzymes in food are extremely low and that the potential for sensitization and allergic reactions in consumers is virtually zero. DSM also reports that they conducted an amino acid sequence homology search between lipase and known allergens included in publicly available allergen databases. DSM concludes that no meaningful homology was detected and that this result supports the conclusion that lipase is not a potential food allergen.
Section 301(ll) of the Federal Food, Drug, and Cosmetic Act (FFDCA)
Section 301(ll) of the FFDCA prohibits the introduction or delivery for introduction into interstate commerce of any food that contains a drug approved under section 505 of the FFDCA, 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 DSM’s notice that the lipase enzyme preparation is GRAS for use in baked goods, FDA did not consider whether section 301(ll) or any of its exemptions apply to foods containing the lipase enzyme preparation. Accordingly, this response should not be construed to be a statement that foods that contain the lipase enzyme preparation, if introduced or delivered for introduction into interstate commerce, would not violate section 301(ll).
Based on the information provided by DSM, as well as other information available to FDA, the agency has no questions at this time regarding DSM’s conclusion that the lipase 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 the lipase enzyme preparation. As always, it is the continuing responsibility of DSM 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 000296, 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 via the FDA home page at http://www.fda.gov. To view or obtain an electronic copy of the text of the letter, follow the hyperlinks from the “Food” topic to the “Food Ingredients and Packaging” section to the “Generally Recognized as Safe (GRAS)” page where the GRAS Inventory is listed.
Mitchell A. Cheeseman, Ph.D.
Office of Food Additive Safety
Center for Food Safety and Applied Nutrition