Agency Response Letter
GRAS Notice No. GRN 000205

Ms. Lori Gregg
Novozymes North America, Inc.
77 Perry Chapel Church Road
P.O. Box 576
Franklinton, NC 27525

Re: GRAS Notice No. GRN 000205

Dear Ms. Gregg:

The Food and Drug Administration (FDA) is responding to the notice, dated June 5, 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 this notice on June 7, 2006, filed it on June 9, 2006, and designated it as GRAS Notice No. GRN 000205.

The subject of the notice is pullulanase enzyme preparation from Bacillus subtilis expressing the pullulanase gene from B. acidopullulyticus (pullulanase enzyme preparation). The notice informs FDA of the view of Novozymes North America, Inc. (Novozymes) that pullulanase enzyme preparation is GRAS, through scientific procedures, for use in the beer brewing industry as an enzyme for the saccharification of liquefied starch. The pullulanase enzyme preparation is intended for use at minimum levels necessary to achieve the desired effect. Novozymes states that the maximum recommended use level of pullulanase enzyme preparation is 25 liters per ton of starch dry substance. The enzyme preparation is added at the mashing stage. The enzyme activity will be completely inactivated during wort boiling and the protein will be removed in subsequent processing steps.

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. Novozymes' notice provides information about each of the components of the pullulanase enzyme preparation.

Novozymes provides general information about the identity and technical effect of pullulanases as well as specific information about the pullulanase enzyme preparation that is the subject of GRN 000205. It is identified by IUB name alpha-dextrin endo-1, 6-alpha-glucosidase, EC No. 3.2.1.41, and Chemical Abstract Service Registry No. 9075-68-7. Pullulanase catalyzes the hydrolysis of 1,6-alpha-glucosidic linkages in polysaccharides such as pullulan, amylopectin, and glycogen. It is therefore well suited for "debranching" of starch after liquefaction.

The use of pullulanase enzyme in addition to glucoamylase enzyme will speed up the hydrolysis of branched dextrins and will increase the level of fermentable sugars in the wort. The enzymes are subsequently heat inactivated and removed by filtration.

In assessing of the safety of the production organism, B. subtilis, Novozymes 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. Novozymes states that B. subtilis has a history of safe use in food and is considered to be safe for the production of enzymes or ingredients for use in food as evidenced by scientific publications and by several expert groups. The production organism B. subtilis complies with the Organization for Economic Co-operation and Development criteria for Good Industrial Large Scale Practice microorganisms. The notifier also cites a published article that concludes that B. subtilis meets the criteria for a safe production microorganism.

Novozymes states that the donor for the pullulanase enzyme gene, B. acidopullulyticus, has been used as the production organism for a pullulanase enzyme preparation (the subject of GRAS Petition 4G0295) that has been marketed in the United States since 1985. Based on published and unpublished information, Novozymes concludes that B. acidopullulyticus is neither pathogenic nor toxigenic. Novozymes asserts that pullulanases from genetically engineered production organisms have also been marketed in the United States and have a long history of safe use in the food industry.

In assessing the safety evaluation of food components derived from genetically engineered organisms, Novozymes states that the genetic modifications of the production strain are well characterized and specific and do not encode and express any known harmful or toxic substances. Novozymes also assessed the identity and stability of the integrated DNA using Southern blot hybridization and concluded that the DNA is stably integrated into the B. subtilis chromosome.

In its notice, Novozymes cites published comprehensive reviews and studies that support the safety of microbial enzyme preparations used in food processing. Novozymes also presents data from an unpublished 13-week subchronic oral toxicity study in rats, one genotoxicity study (an in vitro bacterial reverse mutation assay), and one in vitro quantitative cytotoxicity test. Novozymes concludes that these tests show that the pullulanase enzyme preparation does not exhibit mutagenic activity or toxic effects under the specified testing conditions.

Novozymes states that the raw materials used in the fermentation and recovery process for the pullulanase enzyme concentrate are common ingredients used in the enzyme industry. Additionally, Novozymes states that the raw materials are suitable food grade materials. Novozymes affirms that the quality management system used in the manufacturing process for the enzyme preparation complies with the requirements of ISO 9001. The enzyme preparation is produced following good manufacturing practices (GMPs).

The pullulanase enzyme preparation is produced by submerged fed-batch pure culture fermentation of the genetically modified strain of B. subtilis. During all steps of fermentation, physical, and chemical control measures are taken and microbiological analyses are performed to ensure the absence of foreign microorganisms and to confirm the identity of the production strain. Novozymes states that its pullulanase enzyme preparation complies with the purity criteria recommended for enzyme preparations as described in the Food Chemical Codex, 5th Edition, 2003 and conforms to the general specifications outlined by the Joint Food and Agriculture Organization/World Health Organization Expert Committee on Food Additives.

Based on the information provided by Novozymes, as well as other information available to FDA, the agency has no questions at this time regarding Novozymes' conclusion that pullulanase 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 pullulanase enzyme preparation. As always, it is the continuing responsibility of Novozymes 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 000205, 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