Agency Response Letter
GRAS Notice No. GRN 000212

Hitoshi Morimoto
Director, Bio/Fine Chemicals Division
Nagase ChemteX Corporation
1-1-17, Shinmachi
Nishi-ku
Osaka 550-8668
JAPAN

Re: GRAS Notice No. GRN 000212

Dear Mr. Morimoto:

The Food and Drug Administration (FDA) is responding to the notice, dated August 17, 2006, 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 August 31, 2006, filed it on September 5, 2006, and designated it as GRAS Notice No. GRN 000212.

The subject of the notice is phospholipase A2 (PLA₂) enzyme preparation from Streptomyces violaceoruber expressing a gene encoding PLA₂ from the same species. This letter refers to the subject of the notice as "S. violaceoruber PLA₂ enzyme preparation." The notice informs FDA of the view of Nagase ChemteX Corporation (Nagase ChemteX) that S. violaceoruber PLA₂ enzyme preparation is GRAS, through scientific procedures, for use as an enzyme to hydrolyze soy lecithin and egg yolk lecithin.

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. Nagase ChemteX's notice provides information about each of these components of the S. violaceoruber PLA₂ enzyme preparation.

Nagase ChemteX provides general information about PLA₂: it is classified by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (formerly Enzyme Commission) Number E.C. 3.1.1.4 and Chemical Abstracts Service Registry Number 9001-84-7. The systematic name of PLA₂ is phosphatidylcholine 2-acylhydrolase. Nagase ChemteX notes that PLA₂ catalyzes the hydrolysis of the ester bond between glycerol and the fatty acid in the.number two position of the glycerol backbone of diacylphospholipids such as lecithin. Hydrolysis of lecithin by PLA₂ yields one molecule of fatty acid (from the sn-2 position of the glycerol moiety) and one molecule of lysolecithin. After incubation with egg yolk or soy lecithin, the enzyme is either inactivated by heat treatment or removed by further purification processes. The resulting modified lecithin has improved emulsifying properties.

Nagase ChemteX notes that the amino acid sequence of the enzyme produced by the production strain of S. violaceoruber (designated AS-10) is identical to that of the donor strain S. violaceoruber IFO 15146. The notifier cites scientific reviews 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. Based on these publications and recommendations, the notifier concludes that a safety assessment of an enzyme preparation derived from a recombinant production organism includes identifying and characterizing both the recipient strain (S. violaceoruber 1326)and the introduced genetic sequences (which includes vector and donor DNA). The notifier provides information about the production microorganism (S. violaceoruber AS-10) that is used as the source of S. violaceoruber PLA₂ enzyme preparation. Nagase ChemteX notes that the enzyme in this enzyme preparation is functionally the same as that in the enzyme preparation that is the subject of GRAS Notice No. GRN 000145, submitted by Genencor.

Nagase ChemteX relies on published reviews for its view that the safety of the production organism is the prime consideration in assessing the probable degree of safety of an enzyme preparation intended for use in food and notes that the recipient organism, designated S. violaceoruber 1326, is nontoxic, nonpathogenic, well-known and well-characterized.¹ Its use meets Organisation for Economic Cooperation and Development criteria for Good Industrial Large Scale Practice. The expression plasmid used to transform S. violaceoruber 1326 consists of the structural gene for PLA₂ from S. violaceoruber IFO 15146, a promoter and terminator of the pld gene (encoding phospholipase D) from S. cinnamoneum, and synthetic linker sequences, as well as a large segment from a streptomyces cloning vector. Nagase ChemteX notes that no homology with known bacterial toxins was found in the complete DNA sequence of the plasmid. In addition, no unintended open reading frames that would encode any known toxins were found. The production strain, designated S. violaceoruber AS-10 contains the expression plasmid as an autonomously replicating extrachromosomal element.

Nagase ChemteX notes that the manufacturing process for its S. violaceoruber PLA₂ enzyme preparation follows standard industry practices described in published reviews and that the enzyme preparation is produced in accordance with good manufacturing practices. Nagase ChemteX notes that standard ingredients used in the enzyme industry are used for fermentation and recovery of the enzyme. Nagase ChemteX produces the enzyme from a submerged fed-batch pure culture fermentation of the S. violaceoruber production strain, carried out using physical and chemical measures and microbial analysis to ensure the absence of foreign microorganisms. Following fermentation, the recovery process includes a primary vacuum drum filtration, precipitation with ammonium sulfate, re-suspension in water, a second vacuum drum filtration, further filtration to eliminate any remaining production strain organisms, and preservation and stabilization of the liquid enzyme concentrate. Nagase ChemteX blends the stabilized concentrate with water and sorbitol and preserves it with potassium sorbate and salt.

Nagase ChemteX markets the PLA₂ enzyme preparation in a formula for use in producing enzyme-modified lecithin (from soy or egg yolk). Its typical composition includes about one percent total organic solids (TOS) and nine percent ash. The enzyme preparation complies with the general and additional requirements for enzyme preparations set forth in the Food Chemicals Codex (4th edition, 3rd supplement, 2001) and to the general specifications for enzyme preparations used in food processing provided by the Joint FAO/WHO Expert Committee on Food Additives (2001). Nagase ChemteX notes a typical activity of 10,000 PLA₂UN² per milliliter (ml).

Nagase ChemteX intends its S. violaceoruber PLA₂ enzyme preparation to be used at levels up to 20 PLA₂UN per gram egg yolk (equivalent to 0.02 mg TOS per gram egg yolk) and 200 PLA₂UN per gram of lecithin (or 0.2 mg TOS per gram lecithin). Nagase ChemteX estimates exposure based on conservative assumptions (that all soy lecithin is enzyme-modified, all PLA₂ added remains in the modified lecithin and in the final food (e.g., mayonnaise) prepared with modified lecithin). Nagase Chemtex provides an exposure estimate of 0.164 mg TOS per person per day (equivalent to 0.0027 mg TOS per kilogram body weight per day). However, Nagase ChemteX notes that the PLA₂ enzyme is largely removed from modified soy lecithin by further purification processes.

Nagase ChemteX summarizes the results of unpublished studies performed on a concentrated S. violaceoruber PLA₂ enzyme preparation, including in vitro reverse mutation and chromosome aberration assays, and, in rats, acute oral LD50 assays and a 13-week oral toxicity study. Nagase ChemteX concludes that the enzyme preparation is noncytotoxic and nonmutagenic in bacterial or mammalian cells under the study conditions. The notifier reports that the 13-week oral toxicity study resulted in a no observed adverse effect level of 200 mg/kg bw/day for male rats and 40 mg/kg bw/day for female rats. Nagase ChemteX concludes that the data is sufficient to preclude toxicity of the enzyme preparation.

Based on the information provided by Nagase ChemteX, as well as other information available to FDA, the agency has no questions at this time regarding Nagase ChemteX's conclusion that S. violaceoruberPLA₂ 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 S. violaceoruber PLA₂ enzyme preparation. As always, it is the continuing responsibility of Nagase ChemteX 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 000212, 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

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⁽¹⁾Publicly available information about this donor strain establishes its identity with the long-established laboratory strain known as S. coelicolor or S. violaceoruber A3(2).

⁽²⁾PLA₂UN is defined as the amount of enzyme producing 1 microgram (µg) free fatty acid per minute when the enzyme is reacted at 37 degrees Celsius, pH 8.0, with a one percent L-alpha-phosphatidylcholine solution.