June 12, 2001

Gary L. Yingling
Kirkpatrick & Lockhart, L.L.P.
1800 Massachusetts Avenue
Washington, D.C. 20036

Re: GRAS Notice No. GRN 000072

Dear Mr. Yingling:

The Food and Drug Administration (FDA) is responding to the letter, dated March 9, 2001, that you submitted on behalf of Genencor International, Inc. (Genencor). Your letter requests that FDA convert the filed GRAS affirmation petition GRP 5G0415 to a GRAS notice 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). Although GRP 5G0415 was submitted to FDA by a different firm (i.e., Solvay Enzymes, Inc.), Genencor acquired all rights and interests in GRP 5G0415 on July 1, 1996.

FDA received Genencor's conversion request on March 10, 2001 and designated it as GRAS Notice No. GRN 000072. The subject of the conversion request is pullulanase enzyme preparation obtained from a strain of Bacillus licheniformis that contains a gene encoding pullulanase derived from B. deramificans. The conversion request informs FDA of the view of Genencor that this pullulanase enzyme preparation is GRAS, through scientific procedures, for use as a processing aid in the manufacturing of starch hydrolysates (maltodextrins, maltose, glucose, and high fructose corn syrup (HFCS)). The pullulanase enzyme preparation would be used in starch saccharification at a level of 2.3 milligrams of enzyme protein per kilogram of starch (i.e., 2.3 parts per million), in accordance with current Good Manufacturing Practices (cGMPs). Because starch hydrolysates are subjected to repeated purification steps, Genencor expects that the level of the pullulanase enzyme preparation in finished food ingredients derived from treated starch will be a small fraction of its initial concentration in liquefied starch. Genencor estimates that the enzyme residue in glucose syrup and crystalline glucose will not exceed 23 parts per billion and that the enzyme residue in HFCS will not exceed 0.23 parts per billion.

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 that derive from the source organism and constituents that derive from the manufacturing process, e.g., components of the fermentation media or the residues of processing aids. GRP 5G0415 provides information about each of these components of pullulanase enzyme preparation.

The systematic name of pullulanase (EC No.3.2.1.41) is pullulan 6-glucanohydrolase. GRP 5G0415 describes published information about the technical effect of pullulanase, including the substrates for pullulanase (i.e., amylopectin and pullulan). Amylopectin is a highly branched polysaccharide that is a principal component of edible starch. Amylopectin consists of linear polymers of 1,4-alpha-linked D-glucose units joined at branch points by 1,6-alpha-glucosidic linkages. Pullulan is a linear polysaccharide composed of maltotriose units linked by alpha-D-(1,6) bonds. During the process known as "wet milling," corn starch is hydrolyzed using the enzyme glucoamylase, which catalyzes the stepwise hydrolysis of alpha-1,4-linkages in starch. Although glucoamylase also is capable of hydrolyzing the alpha-1,6- linkages, this reaction proceeds much more slowly.

Pullulanase is used in the wet milling of corn starch, during the saccharification with glucoamylase, to specifically cleave the 1,6-alpha-glucosidic linkages in amylopectin and pullulan. This use of pullulanase reduces the level of glucoamylase that is used during saccharification, increases the ultimate glucose yield, allows the saccharification process to be carried out at higher levels of dissolved solids, and shortens the saccharification time.

GRP 5G0415 describes scientific publications and recommendations, issued by FDA or by international organizations, about the safety of enzymes used in food processing, including enzymes derived from microorganisms that are modified using recombinant deoxyribonucleic acid (rDNA) technology. Consistent with those documents, GRP 5G0415 contains published information pertaining to the host organism, the various components of the plasmids that were introduced into the host organism, the production organism, and the characteristic properties of enzyme component.

GRP 5G0415 contains published information pertaining to the host organism, B. licheniformis strain SE2 delap1, which is used in the construction of the production strain (B. licheniformis strain SE2-pul-int211).⁽¹⁾ B. licheniformis strains are common in most soils and are listed in the Food Chemicals Codex as a source of carbohydrase and protease enzyme preparations used in food processing. The FDA has affirmed that a mixed carbohydrase and protease enzyme product derived from B. licheniformis is GRAS for use in the production of certain foods (21 CFR 184.1027). GRP 5G0415 cites published reports on the cloning and expression of proteins in B. licheniformis for use in food products. Genencor concludes that common B. licheniformis strains are non-pathogenic to humans, animals, and plants.

GRP 5G0415 contains published information pertaining to the various components of the plasmids that were introduced into B. licheniformis strain SE2 delap1. The gene that encodes pullulanase derives from another species of Bacillus, B. deramificans. GRP 5G0415 describes the cloning process (which employed standard, well-known vectors that do not raise any safety concerns), and describes published and unpublished information pertaining to the production strain (B. licheniformis strain SE2-pul-int211). GRP 5G0415 includes data and information derived using the technique of Southern hybridization to demonstrate that the DNA sequence encoding pullulanase is stably integrated into the B. licheniformis host chromosome. GRP 5G0415 also describes an experiment analyzing the transformation potential of the recombinant production strain using competent E. coli cells and total DNA extracted from the production strain. Genencor concludes that the experiment shows that there is no transformation potential from the integrated vector.

GRP 5G0415 includes a published article that describes a study conducted in rats to determine the pathogenic potential of the production strain. In this study, groups of 5 male and 5 female rats received one-time intraperitoneal injections of either live or killed cells in doses of 10⁶, 10⁹, and 10¹¹ bacteria per kilogram of body weight. No animals demonstrated signs of pathogenicity at any dose level of either live or killed cells. Genencor concludes that the production strain is, as expected, non-pathogenic in rats.

GRP 5G0415 describes the manufacturing process for pullulanase enzyme preparation. Pullulanase enzyme preparation is produced by a submerged, aerobic, and pure culture fermentation of B. licheniformis strain SE2-pul-int211 in accordance with cGMP. The manufacturing process consists of fermentation, recovery, and formulation. During the fermentation process, propagation is carried out on a small scale to ensure that the bacilli are viable and are a pure culture. The culture is transferred to a primary fermentor for seed fermentation, which generates an initial biomass. The biomass from the seed fermentation is then used in the main fermentation to generate large quantities of biomass. During the recovery process, the enzyme is separated from the fermentation debris and purified and concentrated by ultrafiltration, which ensures that the concentrated enzyme solution is free of the production strain and insoluble components from the fermentation medium. During the formulation process, the enzyme concentrate is stabilized with potassium sorbate and sodium benzoate and standardized at 40 percent solids with corn syrup. Pullulanase enzyme preparation complies with the general and additional requirements for enzyme preparations set forth in the Food Chemicals Codex (4th ed., 1996).

GRP 5G0415 includes data and information derived from DNA sequencing to demonstrate that the DNA sequence encoding pullulanase is the same as that derived from B. deramificans. GRP 5G0415 compares the pullulanase produced by the recombinant B. licheniformis with that obtained from B. deramificans. The analyses included determination of molecular weight, optimal pH and optimal temperature for pullulan hydrolysis, ion exchange chromatography profile, pattern of hydrolysis products, and amino-terminal sequencing. Genencor concludes that the pullulanase produced by the recombinant production strain and the pullulanase produced by the donor strain are essentially the same.

GRP 5G0415 describes a published article describing toxicity studies performed with the pullulanase enzyme preparation derived from the production strain. These studies include a 14-day feeding study in rats, a 28-day feeding study in rats, a bacterial reverse mutation assay in Salmonella typhimurium (Ames test), an in vitro histidine forward mutation assay in mouse lymphoma cells, in vivo mouse bone marrow micronucleus and chromosomal aberration assays, an acute inhalation toxicity study, and primary dermal irritation studies. The maximum concentration of the pullulanase enzyme preparation used in each of the four genetic toxicity studies showed no indication of mutagenicity or genotoxic activity. The highest dose level used in the 14-day and the 28-day feeding studies (5 percent of the diet) revealed no signs of toxic effects. Genencor concludes that pullulanase enzyme preparation derived from the production strain is, as expected, non-mutagenic and non-toxigenic.

FDA has evaluated the information provided by Genencor, as well as the information in GRP 5G0415 and other information available to the agency. Based on this evaluation, the agency has no questions at this time regarding Genencor's conclusion that pullulanase enzyme preparation obtained from a strain of B. licheniformis, which contains a gene encoding pullulanase derived from B. deramificans, 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 pullulanase 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 the interim policy discussed in the GRAS proposal (62 FR 18938 at 18954), FDA has not committed any resources to review of GRP 5G0415 since March 10, 2001, the date that we received your conversion request. At this time, we request that you formally withdraw GRP 5G0415.

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 the notice that conforms to the information in proposed 21 CFR 170.36(c)(1), is available for public review and copying on the Office of Premarket Approval's homepage on the Internet (at http://www.cfsan.fda.gov/~lrd/foodadd.html).

⁽¹⁾The host organism derives from the B. licheniformis strain SE2. Although B. licheniformis strain SE2 contains a gene encoding alkaline protease, this alkaline protease gene has been deleted from B. licheniformis strain SE2 delap1.

^* The Office of Premarket Approval became the Office of Food Additive Safety on June 18, 2001.

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