Agency Response Letter GRAS Notice No. GRN 000277
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CFSAN/Office of Food Additive Safety
May 15, 2009
Alice J. Caddow
Genencor, a Danisco Division
925 Page Mill Road
Palo Alto, CA 94304
Re: GRAS Notice No. GRN 000277
Dear Ms. Caddow:
The Food and Drug Administration (FDA) is responding to the notice, dated January 7, 2009, 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 January 9, 2009, filed it on January 13, 2009, and designated it as GRAS Notice No. GRN 000277.
The subject of the notice is a maltotetraohydrolase enzyme preparation from Bacillus licheniformis expressing a modified maltotetraohydrolase gene from Pseudomonas stutzeri (G4-amylase enzyme preparation). The notice informs FDA of the view of Genencor, a Danisco Division (Genencor), that the G4-amylase enzyme preparation is GRAS, through scientific procedures, for use as an enzyme in bread and other baked goods to delay the staling process.
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, Genencor provides information about all the components of the G4-amylase enzyme preparation.
G4-amylase is a maltotetraohydrolase that catalyzes the hydrolysis of 1,4-α-D-glucosidic linkages in maltotetraose polysaccharides to remove successive maltotetraose residues from the non-reducing chain ends. According to the classification of enzymes established by the International Union of Biochemistry and Molecular Biology, maltotetraohydrolase is identified by the Enzyme Commission number 184.108.40.206. Its accepted name is glucan 1,4-α-D-maltotetraohydrolase and its systematic name is 4-α-D-glucan maltotetraohydrolase. The Chemical Abstract Service Registry number of maltotetraohydrolase is 37288-44-1. Genencor notes that the G4-amylase is a variant of the wild type maltotetraohydrolase from P. stutzeri, which was modified by removing the starch-binding domain and changing 16 out of the remaining 429 amino acids to improve its thermostability and baking performance.
Genencor describes the host microorganism, B. licheniformis strain BRA7, as derived from a B. licheniformis strain lineage that has been used in the production of several commercial enzymes used in food processing including α-amylase, protease, pullulanase, and xylanase. Genencor states that B. licheniformis is nonpathogenic and nontoxigenic, meets the criteria for a safe production microorganism described by several expert groups, and is widely used by enzyme manufacturers for the production of enzyme preparations for use in human food.
Genencor describes the development of the host and production strains. The host strain BRA7 was modified by deletion of several enzyme activities (proteases and amylase), a sporulation gene, and the native chloramphenicol resistance gene (the cat gene). The strain was subsequently transformed with a plasmid vector carrying the expression cassette with the modified maltotetraohydrolase gene (G4-amylase gene) under the control of B. licheniformis promoter and terminator and the native B. licheniformis cat gene. The cat gene was used for selection, chromosomal integration, and amplification of the expression cassette. After integration, all vector sequences were deleted. Genencor states that since the native B. licheniformis cat gene was utilized in the construction of the production strain, no new antibiotic resistance was introduced. Genencor also notes that the production strain was evaluated to confirm that it contains only the intended genetic modifications.
Genencor describes the manufacture of the G4-amylase using submerged straight-batch or fed-batch pure culture fermentation of the genetically modified strain of B. licheniformis. Each production batch is initiated from a lyophilized stock culture, which is tested for identity, absence of foreign microorganisms and enzyme generating ability before use. All fermentation steps are conducted under controlled conditions and monitored for microbial contamination. The G4-amylase is secreted to the fermentation broth and is subsequently recovered via several purification and concentration steps. The resulting liquid concentrate is stabilized with potassium sorbate and tested to demonstrate that it meets the intended specifications. The concentrate is subsequently mixed with microcrystalline cellulose and spray dried. The final products are formulated either as powder blends or tablets using microcrystalline cellulose and other ingredients, such as wheat starch, sodium chloride, calcium carbonate, citric acid, or BHT. The total organic solids (TOS) content of the final G4-amylase enzyme preparations is approximately 9.1%.
Genencor states that enzyme products conform to the general and additional requirements for enzyme preparations as 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.
Genencor states that the G4-amylase is intended for use in bakery products to delay staling by shortening the amylopectin side chains and releasing maltooligosaccharides. The recommended use levels range from 2 to 30 milligrams (mg) enzyme protein per kilogram (kg) flour (equivalent to 2 to 34 mg TOS per kg flour). The enzyme is expected to be inactivated during baking. Genencor estimates a maximum daily intake of the G4-amylase to be 0.098 mg of the enzyme protein per kilogram of body weight per day (mg/kg bw/d; equivalent to 0.11 mg TOS/kg bw/d) under the assumption that 100% of the enzyme added to flour would remain in baked goods.
Genencor summarizes unpublished toxicological studies conducted with the G4-amylase enzyme which include acute and subchronic (13-week) oral toxicity study in rats, bacterial reverse mutation assay (Ames test), and in vitro mammalian chromosomal aberration test. Genencor states that the enzyme was not toxic in the acute toxicity study and that no systemic toxicity was observed in the subchronic study at the highest dose tested of 90 mg TOS/kg bw/d. Genencor also concludes that the genotoxicity studies demonstrated no mutagenic, clastogenic, or aneugenic effects.
Genencor provides general information regarding potential allergenicity of enzymes used in food processing. Genencor states that enzyme dust can lead to dermal and respiratory sensitization among industry workers but the rate of such occurrences is relatively low. Genecor discusses published studies which show that ingestion of small amounts of enzymes present in food generally does not induce allergic reactions in consumers even in individuals with inhalation allergy to enzymes. In addition, Genencor compares the amino acid sequence of the G4-amylase to the sequences of known allergens contained in two allergen databases and concludes that the enzyme does not match the amino acid sequences of any known food allergens when tested according to the FAO/WHO (2001) recommendations.
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 Genencor’s notice that the G4-amylase enzyme preparation is GRAS for use in bread and other baked goods to delay the staling process, FDA did not consider whether section 301(ll) or any of its exemptions apply to foods containing the G4-amylase enzyme preparation. Accordingly, this response should not be construed to be a statement that foods that contain the G4-amylase enzyme preparation, if introduced or delivered for introduction into interstate commerce, would not violate section 301(ll).
Based on the information provided by Genencor, as well as other information available to FDA, the agency has no questions at this time regarding Genencor’s conclusion that the G4-amylase 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 G4-amylase enzyme preparation. As always, it is the continuing responsibility of Genencor 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 000277, 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.
Laura M. Tarantino, Ph.D.
Office of Food Additive Safety
Center for Food Safety and Applied Nutrition