Agency Response Letter
GRAS Notice No. GRN 000145

Mr. Gary Yingling
Kirkpatrick & Lockhart LLP
1800 Massachusetts Avenue, NW
Suite 200
Washington, DC 20036-1221

Re: GRAS Notice No. GRN 000145

Dear Mr. Yingling:

The Food and Drug Administration (FDA) is responding to the notice, dated February 6, 2004, that you submitted on behalf of Genencor International, (Genencor, the notifier) 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 9, 2004, filed it on February 11, 2004, and designated it as GRAS Notice No. GRN 000145.

The subject of the notice is phospholipase A2 (PLA2) enzyme preparation from Streptomyces violaceoruber (PLA2 enzyme preparation⁽¹⁾). The notice informs FDA of the view of Genencor that PLA2 enzyme preparation from S. violaceoruber is GRAS, through scientific procedures, for use as an enzyme for the hydrolysis of phospholipids in soy lecithin and egg yolk lecithin at levels not to exceed good manufacturing practices. Genencor estimates that typical use levels are 0.5-5 units of enzyme per gram (U/g) of lecithin. Genencor states that human consumption of the enzyme is expected to be negligible, because the concentrations of enzyme required to achieve the intended technical effect are low, and the enzyme often is removed and/or denatured in the final food product.

Commercial enzyme preparations that are used in food processing 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 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 each of these components of the PLA2 enzyme preparation from S. violaceoruber.

PLA2 belongs to the class of enzymes called lipases. Genencor discusses the safe use of lipases in food processing. The notice cites use of microbial lipases since at least 1952, the presence of PLA2 enzymes in all cells, and discusses a study that indicated that enzyme-modified lecithin, the PLA2-hydrolyzed product, posed no toxiciological hazards over untreated lecithin. Genencor also provides the following information:

• FDA has affirmed the following lipases as GRAS for their intended uses:
  • Animal lipases from edible forestomach tissue of calves, kids, lambs, or from animal pancreatic tissue (21 CFR 184.1415) and lipase enzyme preparation from Rhizopus niveus (21 CFR 184.20)
  • FDA approved a food additive regulation for esterase-lipase derived from Mucor miehei (21 CFR 173.140)
• Other lipase enzyme preparations have been the subjects of GRAS notices from the following sources: Penicillium camembertii (GRN No. 0000068), Aspergillus niger (GRN No. 000111), Aspergillus oryzae (GRN No. 000113), and several preparations in which various enzymes are produced in A. oryzae (GRN 000043, 75, 103)
• A publication by Pariza and Johnson (2001) on the safety of microbial enzymes including PLA2 enzyme preparation from S. violaceoruber.

Genencor describes published information about the technical effects of PLA2 enzymes, also known as lecithinases, a category of lipases that catalyzes the hydrolysis of the ester bond between glycerol and the fatty acid in the number two position of the glycerol backbone of lecithin.⁽²⁾ PLA2 thus produces a modified lecithin with improved emulsifying ability and a free fatty acid. Genencor provides generally available information about the chemical identity of PLA2: the systematic name of PLA2 is phosphatidylcholine 2-acylhydrolase, the Enzyme Commission number is 3.1.1.4 (EC 3.1.1.4), and the Chemical Abstracts Service Registry Number (CAS Reg. No.) is 9001-84-7. Genencor cites published information characterizing PLA2 from S. violaceoruber in which its alpha-helical structure and number of cysteine residues (four) distinguish it from eucaryotic PLA2 enzymes, which have beta-sheet and alpha-helical domains and a greater number (12-16 total) of cysteine residues.

Genencor cites scientific review articles in support of its view that the safety of the production organism is the prime consideration in assessing the safety of an enzyme preparation intended for use in food. Additionally, Genencor cites a publication of the International Food Biotechnology Council, which concludes that if the production microorganism is nontoxigenic and nonpathogenic and the manufacturing process is conducted using current good manufacturing practices (cGMPs), the food or food ingredient produced from that microorganism is safe to consume. Genencor provides publicly available information including taxonomic information, in support of its conclusion that S. violaceoruber is considered nonpathogenic and nontoxigenic. Genencor mentions a publication that reported that a strain of S. violaceoruber produced aflatoxin B2, but refutes this report by noting that only fungi (not bacteria such as S. violaceoruber) are known to produce aflatoxins. Genencor describes a test it characterizes as a test for pathogenicity and toxicity; neither toxicity nor death resulted when viable cells were administered to mice by gavage or by intraperitoneal injection from 0.00025 to 5000 milligrams per kilogram body weight (mg/kg bw).

Genencor states that PLA2 enzyme preparation from S. violaceoruber is manufactured using commonly accepted methods for the manufacture and formulation of enzymes and that the enzyme preparation is produced according to cGMPs. Genencor states that the raw materials and processing aids used in fermentation and recovery processes are standard materials used by the enzyme industry and are suitable for general use in food. The PLA2 enzyme is produced by a pure submerged culture fermentation of S. violaceoruber. The fermentation conditions are monitored, and factors such as pH, temperature, and aeration are controlled. The culture is periodically tested for microbiological contamination, and any contaminated fermentation is rejected. Following fermentation, a multi-step recovery process is performed, which includes purification and concentration of the product. The enzyme preparation is formulated as a liquid product, and is standardized according to product specifications. The total organic solids (TOS) concentration of PLA2 enzyme preparation from S. violaceoruber is 1.0 x 10-4 g/U of PLA2. The enzyme is packaged and stored at 4 degrees Celsius.

Genencor provides specifications for the enzyme preparation that comply with or exceed the requirements for enzyme preparations as set forth in the Food Chemicals Codex (4th Edition, 1996) and by the Joint Expert Committee on Food Additives (JECFA, 2001).

Genencor's notice includes a summary of unpublished studies performed with the PLA2 enzyme preparation from S. violaceoruber, including an acute oral (gavage) LD50 study in rats, a 13-week oral feeding study in rats, a forward mutation assay in mouse lymphoma cells (L5178K cell line), and an unscheduled DNA synthesis assay in rat liver cells. These studies found no treatment-related toxicity, induction of gene mutation or chromosomal aberrations in assays using PLA2 from S. violaceoruber. Based on the 13-week study in rats, Genencor determined a NOEL of PLA2 from S. violaceoruber of 23 mg total protein/kg bw/day(d) (equivalent to 54.8 mg TOS/kg bw/d). Genecor concludes that all these studies demonstrate that PLA2 from S. violaceoruber is non-toxic by ingestion.

Genencor addresses the potential allergenicity of PLA2, citing a scientific review that concludes there have been no confirmed reports of allergies in consumers caused by enzymes used in food processing. Genencor further states that PLA2 from S. violaceoruber is not derived from or related to known food allergens. Hence, Genencor considers risk of allergenicity due to exposure to PLA2 to be negligible.

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 phospholipase A2 enzyme preparation from S. violaceoruber 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 PLA2 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, as well as a copy of the information in your notice that conforms to the information in proposed 21 CFR 170.36(c)(1), is available for public review and copying on the homepage of the Office of Food Additive Safety (on the Internet at http://www.cfsan.fda.gov/~lrd/foodadd.html).

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

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⁽¹⁾PLA2 (EC 3.1.1.4) is generically listed as one of two enzymes used for production of enzyme-modified lecithin, which is affirmed GRAS in 21 CFR 184.1063.

⁽²⁾The term "lecithin" may denote pure phosphatidylcholine, but often is applied commercially to denote a crude mixture of phosphatides that includes phophatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, other phospholipids, triglycerides, fatty acids, carbohydrates, sterols, and glycolipids.