Agency Response Letter GRAS Notice No. GRN 000315
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CFSAN/Office of Food Additive Safety
May 5, 2010
Vincent Sewalt, Ph. D.
Director of Regulatory Affairs
Genencor, a Danisco Division
925 Page Mill Road
Palo Alto, CA 94304
Re: GRAS Notice No. GRN 000315
Dear Dr. Sewalt:
The Food and Drug Administration (FDA) is responding to the notice, dated December 04, 2009, 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 December 7, 2009, filed it on January 6, 2010, and designated it as GRAS Notice No. GRN 000315.
The subject of the notice is a transglucosidase enzyme preparation from Trichoderma reesei expressing the gene encoding transglucosidase from Aspergillus niger. The notice informs FDA of the view of Genencor, a Danisco Division (Genencor) that the transglucosidase enzyme preparation is GRAS, through scientific procedures, for use in the production of isomalto-oligosaccharide syrups from starch and potable ethanol from molasses.
Commercial enzyme preparations that are used in food 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 manufacturing process. In its notice, Genencor provides information about all the components of the transglucosidase enzyme preparation.
Genencor’s transglucosidase catalyzes both the hydrolysis and transfer of D-glucosyl units of oligosaccharides and converts 1,4 glucosidic linkages to 1,6 glucosidic linkages. When used in the production of isomalto-oligosaccharide syrups from starch, transglucosidase converts malto-oligosaccharides to isomalto-oligosaccharides. In molasses, transglucosidase converts non-fermentable sugars such as raffinose and stachyose to sucrose, galactose, glucose and fructose, which can then be fermented into alcohol.
According to the classification system of enzymes established by the International Union of Biochemistry and Molecular Biology (IUBMB), transglucosidase is identified by the Enzyme Commission number 126.96.36.199. Its accepted name is 1,4-α-glucan 6-α- glucosyltransferase, and its systematic name is 1,4-α-D-glucan:1,4-α-D-glucan (D-glucose)-6-α-D-glucosyltransferase. The Chemical Abstract Service Registry number for transglucosidase is 9030-12-0.
Genencor states that the production organism, T. reesei, is non-toxigenic and non-pathogenic and has a long history of safe use in industrial scale enzyme production. In the literature search conducted by Genencor, no reports were found that implicate T. reesei in disease, toxicity, or allergenicity among healthy adults. T. reesei is classified as a Biosafety Level 1 (BSL1) microorganism by the American Type Culture Collection (ATCC). BSL1 microorganisms are not known to cause disease in healthy humans.
According to Genencor, the production strain T. reesei TG #626, GICC 03289 was derived by recombinant DNA methods from T. reesei strain RL-P37. T. reesei strain RL-P37 is a commercial production strain that was derived from the well-known wild-type T. reesei strain QM6a using several classical mutagenesis steps. T. reesei strain RL-P37 has been used by Genencor for production of cellulases for over fifteen years and has been determined to be non-pathogenic and non-toxicogenic via intraperitoneal dosing in rats. Genencor also notes that FDA issued a “no questions” letter in response to GRAS Notice No. 000230 containing information on bovine chymosin obtained from a T. reesei production strain, which was derived from T. reesei strain RL-P37.
Genencor states that the transglucosidase production strain was constructed by transformation of T. reesei strain RL-P37 with the expression vector containing DNA encoding the mature transglucosidase from A. niger and the amdS selectable marker gene from Aspergillus nidulans. The vector was integrated into the chromosomal DNA of the recipient strain and the final construct was verified by Southern blot analysis. The production strain was shown to be stable for at least 60 generations of fermentation, based on transglucosidase production. Genencor states that no antibiotic resistance genes were used in the construction of the production microorganism. Genencor also verified the absence of the production microorganism in the final enzyme product.
Genencor states that the donor organism, A. niger, has a long history of safe use as a source of enzymes and is currently used to produce enzymes, such as protease and glucoamylase. Genencor cites previous use of A. niger transglucosidase in the production of isomalto-oligosaccharides (GRN 000246) as further evidence of its safety.
According to Genencor, transglucosidase is manufactured by submerged straight-batch or fed-batch pure culture fermentation of the T. reesei production strain. The production process is consistent with FDA’s current food good manufacturing practice (cGMP) as set forth in 21 CFR Part 110. Each production batch is initiated from a stock culture, which is tested prior to use for identity, absence of contaminating microorganisms and enzyme generating ability. The fermentation process is conducted under controlled conditions and the fermentation broth is periodically tested for microbial contamination. Transglucosidase is secreted to the fermentation broth and is subsequently recovered via several purification and concentration steps. The resulting liquid concentrate is formulated with sodium chloride and glycerol. The finished enzyme preparation contains approximately 3 percent transglucosidase, 3 percent sodium chloride, 50 percent glycerol and 44 percent water. Genencor states that this product meets the purity specifications for enzyme preparations set forth in the Food Chemicals Codex (6th Edition) and the General Specifications and Considerations for Enzyme Preparations Used in Food Processing established by the FAO/WHO Joint Expert Committee on Food Additives.
Genencor states that the transglucosidase enzyme preparation will be used in the production of isomalto-oligosaccharides at use levels from 0.5 to 1.5 kg per ton of starch dry solids. After two days of reaction, the enzyme is inactivated and removed from the final product via several purification steps. In the production of potable ethanol, the transglucosidase enzyme preparation will be added during fermentation at a level of 6-20 mg/kg. Genencor states that the enzyme will be inactivated and removed from the final potable ethanol product during distillation of the fermentation broth.
To estimate human exposure to the transglucosidase enzyme preparation, Genencor assumed that the enzyme preparation would be used at the highest use level and all would be carried over to the final food product. In such a case, the cumulative exposure to transglucosidase from consumption of both potable ethanol and isomalto-oligosaccharides would be 0.034 mg enzyme per kg body weight per day (mg/kg bw/d) expressed as total protein or 0.040 mg/kg bw/day expressed as total organic solids (TOS), assuming a body weight of 60 kg.
Genencor provided the results of toxicological studies for eight enzyme preparations derived from T. reesei, one from a traditionally modified strain and seven from recombinant strains. Toxicology tests included 91-day subchronic feeding studies in rats for seven enzyme preparations and a 28-day study for one enzyme preparation. The studies also included a bacterial reverse mutation assay and an in vitro chromosomal aberration assay with either human lymphocytes or Chinese hamster ovary cells. All enzyme preparations were found to be non-toxic, non-mutagenic, and not clastogenic. Genencor concludes that the results of these studies as well as pathogenicity studies conducted on T. reesei confirm that T. reesei strain RL-P37 is a safe production host and the enzyme preparations resulting from it are safe for use in food.
Genencor cites published information in support of the notion that the ingestion of food enzymes in general is not considered to be a concern with regard to food allergy. Genencor reports that consistent with the recommendations of the Codex Alimentarius Commission (2003), Genencor conducted the amino acid sequence homology search for transglucosidase from A. niger with known allergens listed in two publicly available allergen databases. Genencor identified no homology of 35% or greater when using an 80-amino acid sliding window and no homology with 8-amino acid stretches. Genencor found one 7-amino acid match and five 6-amino acid matches; however, after analyzing these matches, Genencor eliminated them as potential allergen binding epitopes. Genencor concludes that transglucosidase is unlikely to pose a risk of food allergy.
Section 301(ll) of the Federal Food, Drug, and Cosmetic Act (FFDCA)
Section 301(ll) of the FFDCA prohibits the introduction or delivery for introduction into interstate commerce of any food that contains a drug approved under section 505 of the FFDCA, a biological product licensed under section 351 of the Public Health Service Act, or a drug or a biological product for which substantial clinical investigations have been instituted and their existence made public, unless one of the exemptions in section 301(ll)(1)-(4) applies. In its review of Genencor’s notice that the transglucosidase enzyme preparation is GRAS for use in the production of isomalto-oligosaccharide syrups and potable ethanol, FDA did not consider whether section 301(ll) or any of its exemptions apply to foods containing the transglucosidase enzyme preparation. Accordingly, this response should not be construed to be a statement that foods that contain the transglucosidase enzyme preparation, if introduced or delivered for introduction into interstate commerce, would not violate section 301(ll).
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 the transglucosidase 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 the transglucosidase 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 responding to GRN 000315, as well as a copy of the information in this notice that conforms to the information in the GRAS exemption claim (proposed 21 CFR 170.36(c)(1)), is available for public review and copying via the FDA home page at http://www.fda.gov. To view or obtain an electronic copy of the text of the letter, follow the hyperlinks from the “Food” topic to the “Food Ingredients and Packaging” section to the “Generally Recognized as Safe (GRAS)” page where the GRAS Inventory is listed.
Mitchell A. Cheeseman, Ph.D.
Office of Food Additive Safety
Center for Food Safety and Applied Nutrition