Agency Response Letter
GRAS Notice No. GRN 000230

Alice Caddow
Genencor, A Danisco Division
200 Meridian Centre Boulevard
Rochester, NY 14618-3916

Re: GRAS Notice No. GRN 000230

Dear Ms. Caddow:

In a letter dated February 19, 2008, the Food and Drug Administration (FDA) responded to your notice dated July 20, 2007, 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 July 23, 2007, filed it on July 27, 2007, and designated it as GRAS Notice No. GRN 000230.

In our February 19, 2008, letter, we mistakenly omitted the word "conditions" from a sentence in the penultimate paragraph of the letter. This letter repeats the text of our letter dated February 19, 2008, with the exception of the inclusion of the omitted word. We apologize for any inconvenience or confusion that may have resulted from our mistake.

The subject of the notice is chymosin enzyme preparation from Trichoderma reesei expressing the bovine prochymosin B gene (chymosin enzyme preparation). The notice informs FDA of the view of Genencor, a Danisco Division (Genencor) that chymosin enzyme preparation is GRAS, through scientific procedures, for use as an enzyme in cheesemaking.

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 of the enzyme preparation, and 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. Genencor’s notice provides information about the enzyme component, the production microorganism, and the manufacturing process for chymosin enzyme preparation.

Genencor provides specific information about the identity and activity of chymosin B, the enzyme component of the chymosin enzyme preparation. Genencor notes that chymosin B from T. reesei expressing the bovine prochymosin B gene is identical in amino acid sequence to native bovine chymosin B (specified in the National Center for Biotechnology Information and Swiss- Prot protein sequence databases under accession number P00794).⁽¹⁾ Native chymosin is synthesized in the abomasa⁽²⁾ of unweaned calves, initially as preprochymosin (a precursor form of chymosin that includes a signal secretion sequence and the propeptide). The signal secretion sequence is removed from preprochymosin to yield prochymosin, which is subsequently cleaved at low pH to yield active chymosin. Genencor notes that chymosin B is identified generically by the International Union of Biochemistry and Molecular Biology as chymosin (EC 3.4.23.4), and its Chemical Abstract Service Registry Number is 9001-98-3. Genencor also notes that chymosin is classified as an aspartic endopeptidase and acts by cleaving a single bond between the Phe105 and Met106 residues in the kappa-chain of casein.

Genencor provides information about the regulatory status of enzyme preparations that contain chymosin, noting that many uses are presently described by regulation (21 CFR 184.1685). This regulation describes uses of animal-derived rennet that contains chymosin, as well as microbiallyderived chymosin preparations produced through fermentation. The regulation describes multiple technical effects for these chymosin-containing enzyme preparations in a variety of foods at levels not to exceed good manufacturing practices.

Genencor discusses the evidence of safety for its chymosin enzyme preparation. Genencor first discusses the safety of the production organism and cites published information supporting its conclusion that the safety of the host organism is the prime consideration in assessing the safety of an enzyme preparation intended for use in food. Genencor cites published references that discuss the use of T. reesei for production of food enzymes, noting that it is nontoxigenic and nonpathogenic and has a long history of safe use in industrial scale enzyme production. Genencor indicates that the specific production strain T. reesei GICC03278 was derived by recombinant DNA techniques from the industrial production strain RL-P37. The expression vector used to transform T. reesei RL-P37 includes a synthetic prochymosin B gene, a well-characterized selectable marker from Aspergillus nidulans, a T. reesei chaperonin gene, and T. reesei promoters and terminators. Genencor also notes that the DNA used for transforming the host strain does not contain any antibiotic resistance genes. Genencor states that the T. reesei GICC03278 complies with the Organisation for Economic Co-operation and Development criteria for Good Industrial Large Scale Practice. Genencor also discusses several unpublished toxicity studies that corroborate Genencor’s conclusions regarding the safety of the production organism.

Genencor describes the manufacturing process for its chymosin enzyme preparation, noting that the enzyme is produced by a submerged fed-batch pure culture fermentation of T. reesei strain GICC03278, using standard industry practices for the manufacture of enzymes. Conditions are monitored and equipment maintained to prevent contamination by foreign microorganisms. Genencor notes that following fermentation, a multi-step recovery process is utilized. This process includes the following: cell separation by vacuum drum filtration; concentration by ultrafiltration; precipitation and centrifugation; dissolution and formulation of enzyme preparation; treatment with cellulose; and polish filtration. Genencor states that the raw materials used in fermentation and recovery are standard ingredients used in the enzyme industry for food use.

Genencor states that its specifications for the chymosin enzyme preparation conform to those for enzyme preparations as described in the Food Chemicals Codex (5^th edition) and the general specifications for enzyme preparations used in food processing as proposed by the Joint FAO/WHO Expert Committee on Food Additives. Genencor also provides additional specifications insuring the absence of production organism and mycotoxins in the preparation.

Genencor notes that the recommended use level of its chymosin enzyme preparation in cheesemaking is 50 International Milk Clotting Units (approximately 0.06-0.09 milliliters) of chymosin enzyme preparation per liter of milk. Genencor provides an estimated per capita daily intake of chymosin enzyme preparation of 0.0016 milligrams total organic solids per kilogram of body weight per day. Genencor states that this is a “worst-case” estimate based on several conservative assumptions: (1) the present chymosin enzyme preparation is used in all cheese; (2) no chymosin is removed during the cheesemaking process; and (3) the intake of chymosin enzyme preparation is cumulative (i.e., persons that consume cheese also consume cottage cheese and whey).

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 chymosin enzyme preparation from T. reesei expressing the bovine prochymosin B gene 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 chymosin enzyme preparation from T. reesei expressing the bovine prochymosin B gene. 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 000230, 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 on the homepage of OFAS (on the Internet at http://www.cfsan.fda.gov/~lrd/foodadd.html).

Sincerely,

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

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⁽¹⁾FDA notes that this accession number refers to the sequence for chymosin precursor (preprochymosin or preprorennin) and includes sequences for chymosin A and B, which are both native forms of chymosin and differ only by a single amino acid. The general term “chymosin” may refer to chymosin A or B or both.

⁽²⁾Fourth compartment of the ruminant stomach.