Agency Response Letter
GRAS Notice No. GRN 000204

Mr. Charles L. Morin
Morin & Associates
Suite 1460
388 Market Street
San Francisco, CA 94111

Re: GRAS Notice No. GRN 000204

Dear Mr. Morin:

The Food and Drug Administration (FDA) is responding to the notice, dated May 31, 2006, that you submitted on behalf of Diversa Corporation (Diversa) 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 June 5, 2006, filed it on June 9, 2006, and designated it as GRAS Notice No. GRN 000204.

The subject of the notice is phospholipase C (PLC) enzyme preparation from Pichia pastoris expressing a heterologous phospholipase C gene (PLC enzyme preparation). The notice informs FDA of the view of Diversa that PLC enzyme preparation is GRAS, through scientific procedures, for use as an enzyme in degumming vegetables oils for food use.

As part of its notice, Diversa includes the report of a panel of individuals (Diversa's GRAS panel) who evaluated the data and information that are the basis for Diversa's GRAS determination. Diversa considers the members of its GRAS panel to be qualified by scientific training and experience to evaluate the safety of substances added to food. Diversa's GRAS panel concludes that PLC enzyme preparation, meeting relevant food grade specifications and being produced in accordance with current good manufacturing practice, is GRAS based on scientific procedures under the conditions of its intended use in food.

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. Diversa's notice provides information about the enzyme component, the production microorganism, and the manufacturing process for PLC enzyme preparation. This letter refers to the enzyme component as PLC and the enzyme preparation as PLC enzyme preparation.

Diversa describes published and generally available information about lipases and PLC enzymes, demonstrating that: (1) lipases have a long history of safe use in a variety of food processing applications and are commonly present in foods; (2) PLC catalyzes the hydrolysis of the sn-3 phosphate head group from phospholipids (including phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine) resulting in formation of diacylglycerol and he phosphate ester of the head group; (3) PLC removes phospholipids from food oils, a process called degumming; (4) PLC is a digestive enzyme in the pancreatic juice of mammals including humans; and (5) PLC bears structural similarities to porcine phospholipase A2. Publications also show sequence similarity and immunological cross reaction between the PLC from Bacillus subtilis and the PLC enzyme that is present in the enzyme preparation that is the subject of GRN 000204. Diversa also includes information about the identity of PLC; it is identified by Enzyme Commission (E.C.) No. 3.1.1.4, Chemical Abstracts Service Registry Number (CAS Reg. No.) 9001-86-9, systematic name phosphatidylcholine cholinephosphohydrolase, and alternative names phosphatidylcholine-specific phospholipase, Lecithinase C, and Lipophosphodiesterase I. A molecular weight of 28,141 Daltons and a sequence of 245 amino acids characterize Diversa's PLC enzyme.

In assessing the safety of the production organism, P. pastoris, Diversa relies on 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 food use. Diversa cites the following information to support the safe use of P. pastoris as a source of food ingredients: (1) ATCC classifies P. pastoris as Biosafety Level 1, indicating that it does not cause disease in healthy humans, (2) P. pastoris complies with OECD criteria for Good Industrial Large Scale Practice; (3) it has been used for the production of many proteins including proteins for pharmaceutical use; and (4) it has been reviewed for use in animal feed.

Diversa discusses the DNA added to its P. pastoris production strain. Diversa notes that DNA isolated from an environmental isolate from soil encodes the PLC (referred to as the BD16449 enzyme). Diversa notes that: (1) the BD16449 enzyme encoded by this DNA is similar to the PLC from B. cereus; (2) that the carboxy terminal domain associated with hemolytic activity in some PLC enzymes is absent from the BD16449 enzyme; and (3) that the BD16449 enzyme functions much like other microbial phospholipases. Diversa discusses the other DNA present in its production strain, noting that it obtained the DNA from safe sources such as Saccharomyces cerevisiae, P. pastoris, and synthetic DNA, each with known functions. The bacterial origin of replication and antibiotic resistance gene on the expression vector were removed prior to introduction of the expression vector into P. pastoris. Diversa notes that the expression plasmid containing six copies of the PLC gene is integrated into the P. pastoris AOX1 gene.

Diversa describes the method of manufacture and recovery of its PLC enzyme preparation as a series of standard procedures used in the enzyme industry. Diversa describes the fermentation as a controlled submerged fed-batch pure culture of the P.pastoris production strain, with pH, aeration, enzyme activity, agitation, temperature, and oxygen uptake rate each monitored during fermentation. Diversa discards batches with detectable microbial contamination. After reaching optimal PLC production, the broth is diluted, the cell mass removed, and the remaining fluid filtered. The clarified fluid is ultrafiltered and diafiltered to appropriate salt concentration and pH. Diversa uses food grade stabilizers and diluents to stabilize and standardize the final product. Diversa notes that its PLC enzyme preparation complies with the General and Additional Requirements for Enzyme Preparations in the Fifth Edition of the Food Chemicals Codex and conforms to the General Specifications and Considerations for Enzyme Preparations Used in Food Processing outlined by the Joint FAO/WHO Expert Committee on Food Additives. Diversa provides composition information including total organic solids (TOS) of approximately 5 percent and specifications including enzyme activity, lead content, and microbial levels for PLC enzyme preparation.

Diversa notes that the use level of its PLC enzyme preparation is at levels no higher than necessary to achieve the intended effect, generally 100 to 1000 grams (g) enzyme preparation per metric ton of oil, depending on the oil to be treated and the reaction conditions. Diversa expects very little, if any, residual PLC enzyme preparation to be present in food, because aqueous components such as this enzyme are largely removed during the refining process used for oils degummed using PLC enzyme preparation. For a "worst case scenario" presuming that all of the enzyme remains in the oils, Diversa calculates maximum estimated daily intake of 0.015 mg enzyme/kilogram body weight/day (mg/kg bw/d) or 0.092 mg TOS/kg bw/d assuming average per capita daily consumption of vegetable oils and fats of 90 g/day for a 60 kg person.

Diversa provides information from a publication presenting genotoxicity and acute, subchronic, and inhalational toxicity studies performed on a concentrated PLC enzyme preparation from P. pastoris, prepared without the final formulation step. Diversa describes results from three genotoxicity assays as negative. Diversa also describes an acute oral toxicity study, a 14-day range finding oral toxicity study and a 90 day oral toxicity study, noting no test article-related clinical signs of toxicity. Diversa concludes that the enzyme preparation was neither toxic nor genotoxic.

Based on the information provided by Diversa, as well as other information available to FDA, the agency has no questions at this time regarding Diversa's conclusion that PLC 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 PLC enzyme preparation. As always, it is the continuing responsibility of Diversa 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 000204, 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 the Office of Food Additive Safety (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