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U.S. Department of Health and Human Services

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Agency Response Letter GRAS Notice No. GRN 000265

CFSAN/Office of Food Additive Safety

June 13, 2009

Alice J. Caddow
Genencor, a Danisco Division
925 Page Mill Road
Palo Alto, CA 94304

Re: GRAS Notice No. GRN 000265

Dear Ms. Caddow

The Food and Drug Administration (FDA) is responding to the notice, dated November 3, 2008, 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 November 5, 2008, filed it on November 7, 2008, and designated it as GRAS Notice No. GRN 000265.

The subject of the notice is a glycerophospholipid cholesterol acyltransferase (GCAT) enzyme preparation from Bacillus licheniformis expressing a modified GCAT gene from Aeromonas salmonicida subsp. salmonicida (GCAT enzyme preparation). The notice informs FDA of the view of Genencor, a Danisco Division (Genencor), that the GCAT enzyme preparation is GRAS, through scientific procedures, for use as an enzyme to modify phospholipids to lyso-phospholipids and cholesterol ester in egg yolks to avoid product separation during mayonnaise production; processed meat products for emulsification, better consistency, and reduced cooking loss; cheese for increased yield; eggs to give a softer and more tender crumb in bakery products, and degumming of vegetable oils.

21 CFR 101.4 states that all ingredients must be declared by their common or usual name. In addition, 21 CFR 102.5 outlines general principles to use when establishing common or usual names for nonstandardized foods. Our use of “GCAT enzyme preparation” in this letter should not be considered an endorsement or recommendation of that term as an appropriate common or usual name for the purpose of declaring the substance in the ingredient statement of foods that contain that ingredient. Issues associated with labeling and the appropriate common or usual name of a food are the responsibility of the Office of Nutrition, Labeling and Dietary Supplements in the Center for Food Safety and Applied Nutrition.

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 GCAT enzyme preparation.

As part of its notice, Genencor includes the report of a panel of individuals (Genencor’s GRAS panel) who evaluated the data and information that are the basis for Genencor’s GRAS determination. Genencor considers the members of its GRAS panel to be qualified by scientific training and experience to evaluate the safety of substances added to food. Genencor’s GRAS panel evaluated the safety of the production organism and its lineage, the gene encoding the GCAT enzyme, the manufacturing process, intended uses in food processing, estimates of dietary exposure, unpublished studies, and relevant published literature. Based on this review, Genencor’s GRAS panel concluded that the GCAT enzyme preparation is GRAS under the conditions of its intended use.

GCAT is an acyltransferase that catalyzes the transfer of acyl groups from phospholipids and glycolipids to sterols and other acceptor molecules. According to the classification of enzymes established by the International Union of Biochemistry and Molecular Biology, GCAT is identified by the Enzyme Commission number 2.3.1.43. Its accepted name is phosphatidylcholine-sterol O-acyltransferase and its systematic name is phosphatidylcholine sterol O-acyltransferase. The Chemical Abstract Service Registry number for this enzyme is 9031-14-5. Genencor notes that the amino acid sequence of the GCAT enzyme has been modified from that of the native GCAT enzyme from A. salmonicida subsp. salmonicida in only one amino acid, namely, an aspartic acid substitution for asparagine at position 80. Genencor states that this modification optimizes GCAT expression in B. licheniformis.

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 catH gene). The strain was subsequently transformed with a plasmid vector carrying the expression cassette with the modified GCAT gene under the control of a B. licheniformis promoter and terminator and the native B. licheniformis catH gene. The catH 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 catH gene was used 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 GCAT enzyme using submerged 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 GCAT enzyme is secreted to the fermentation broth and is subsequently recovered via several purification and concentration steps. The resulting liquid concentrate is stabilized with glycerol and potassium sorbate and tested to demonstrate that it meets the intended specifications. Genencor reports that the commercial GCAT enzyme preparation typically contains approximately 83 milligrams (mg) total organic solids (TOS) per gram (mg TOS/g) and that the enzyme preparation will be used according to the current good manufacturing practice at minimum levels necessary to achieve the intended technical effect.

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 Joint FAO/WHO Expert Committee on Food Additives.

Genencor states that the GCAT enzyme preparation is intended for use in egg yolks in the production of mayonnaise, processed meat products, cheese, whole eggs in baked goods, and degumming of vegetable oils. The recommended use levels range from 22.6 to 376.9 milligrams total organic solids per kilogram of food (mg TOS/kg food). The enzyme is expected to be either removed and/or inactivated during the pasteurization or baking of products containing egg yolks or eggs, cooking of processed meat products, and the processing of vegetable oils. Genencor states that residual enzyme in cheese will be active unless the cheese is heated. Genencor estimates a maximum daily intake of the GCAT to be 0.047 mg of the enzyme protein per kg of body weight per day (mg/kg bw/d; equivalent to 0.13 mg TOS/kg bw/d) under the assumption that 100% of the enzyme added to egg yolks, eggs, processed meat products, cheese, and vegetable oils would remain in the final foods.

Genencor summarizes unpublished toxicological studies conducted with the GCAT enzyme which include acute and subchronic (13-week) oral toxicity study in rats, bacterial reverse mutation assay (Ames test), in vitro mammalian chromosomal aberration test, and in vivo mouse micronucleus test. Genencor states that the enzyme was not toxic in the acute toxicity study at the highest dose tested of 600 mg total protein per kg, and that no systemic toxicity was observed in the subchronic study at the highest dose tested of 117 mg TOS/kg bw/d. Genencor also concludes that the genotoxicity studies demonstrated no mutagenic, clastogenic, or aneugenic effects.

Genencor provides general information regarding the potential allergenicity of enzymes used in food processing. Genencor states that exposure to enzymes is very low, and even if they were potentially allergenic when ingested, the likelihood of allergic sensitization of consumers to these proteins is virtually nil. In addition, Genencor compares the amino acid sequence of GCAT 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.

Standards of Identity

In the notice, Genencor states its intention to use GCAT enzyme preparation in several 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.

Use in Meat, Poultry, and Egg Products

During its evaluation of GRN 000265, FDA consulted with the Risk and Innovations Management Division (RIMD), Office of Policy, Program, and Development (OPPD), of the Food Safety and Inspection Service (FSIS) of the United States Department of Agriculture. Under the Federal Meat Inspection Act, the Poultry Products Inspection Act, and the Egg Products Inspection Act, FSIS is responsible for determining the efficacy and suitability of food ingredients in meat, poultry, and egg products as well as prescribing safe conditions of use. Suitability relates to the effectiveness of the ingredient in performing the intended purpose of use and the assurance that the conditions of use will not result in an adulterated product, or one that misleads consumers.

Regarding suitability, FSIS has no objection to the use of GCAT enzyme preparation at a level not to exceed 376.9 mg TOS/kg of total product formulation in products produced under the authority of the Egg Products Inspection Act, including egg yolks, dried egg yolks, and frozen egg yolks. FSIS notes that distributors of the enzyme preparation should notify processors of egg products of the need to review the activation process in order to ensure that a safe food is produced if that egg product is incubated at elevated temperatures for a period of time in order to activate the enzymes. This resulting product is amenable and therefore must be identified and declared on the label as an “egg product” directly followed by the ingredients statement.1

In addition, FSIS has no objection to the use of GCAT enzyme preparation, at a level not to exceed 22.6 mg TOS/kg of total product formulation, as an emulsifier in comminuted meat products.2

The enzyme will need to be listed by its appropriate common or usual name in the ingredients statement on the label of the products in which it is used as an ingredient. Also, the product formula, and their percentages in total, along with a product label, will need to be submitted to the FSIS, Office of Policy and Program Development, Labeling and Program Delivery Division, so that the label can be reviewed and evaluated.

Further questions regarding use in meat, poultry, and egg products should be directed to Dr. John M. Hicks, Jr., RIMD/OPPD/FSIS, via email at john.hicks@fsis.usda.gov or to Dr. Catherine Rockwell, RIMD/OPPD/FSIS, via email at catherine.rockwell@fsis.usda.gov.

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 GCAT enzyme preparation is GRAS for use as an enzyme to modify phospholipids to lyso-phospholipids and cholesterol ester in egg yolks to avoid product separation during mayonnaise production; processed meat products for emulsification, better consistency, and reduced cooking loss; cheese for increased yield; eggs to give a softer and more tender crumb in bakery products, and degumming of vegetable oils, FDA did not consider whether section 301(ll) or any of its exemptions apply to foods containing the GCAT enzyme preparation. Accordingly, this response should not be construed to be a statement that foods that contain the GCAT enzyme preparation, if introduced or delivered for introduction into interstate commerce, would not violate section 301(ll).

Conclusions

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 GCAT 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 GCAT 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 000265, as well as a copy of the information in this notice that conforms to the information in the proposed 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, 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.

Sincerely,

Laura M. Tarantino, Ph.D.
Director
Office of Food Additive Safety
Center for Food Safety and Applied Nutrition


 

cc: John M. Hicks, Jr. DVM, MPH
Risk and Innovations Management Division
Office of Policy and Program Development
Food Safety and Inspection Service
U. S. Department of Agriculture
1400 Independence Avenue, S.W., Room 3549 South
Washington, DC 20250-3700


 



 

1According to FSIS, the term amenable means “accountable or liable to an order or regulation.”

2FSIS notes that comminuted meat products are a subset of processed meat products in which the sizes of meat pieces have been reduced by chopping, flaking, grinding, or mincing, but not by chunking or sectioning. FSIS’s use of the term “comminuted” is based on technical effect data that the notifier submitted for review by FSIS. The data describe the effects of GCAT enzyme preparation on liver sausage, which is a comminuted meat product.