Agency Response Letter GRAS Notice No. GRN 000372
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CFSAN/Office of Food Additive Safety
July 25, 2011
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 000372
Dear Dr. Sewalt:
The Food and Drug Administration (FDA) is responding to the notice, dated January 27, 2011, 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 February 18, 2011, filed it on February 23, 2011, and designated it as GRAS Notice No. GRN 000372.
The subject of the notice is the glucoamylase enzyme preparation from Trichoderma reesei expressing the glucoamylase gene from T. reesei (glucoamylase enzyme preparation). The notice informs FDA of the view of Genencor, a Danisco Division (Genencor) that the glucoamylase enzyme preparation is GRAS, through scientific procedures, for use as an enzyme in grain processing, including the manufacture of corn sweeteners such as high fructose corn syrup, brewing, potable alcohol manufacture, and lactic acid production.
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 glucoamylase enzyme preparation.
The principal activity of Genencor’s glucoamylase is to catalyze the hydrolysis of terminal 1, 4-linked α-D-glucose residues successively from non-reducing ends of carbohydrate chains with release of β-D-glucose.
According to the classification system of enzymes established by the International Union of Biochemistry and Molecular Biology (IUBMB), glucoamylase is identified by the Enzyme Commission number 220.127.116.11. Its accepted name is glucan 1,4-α-glucosidase, and its systematic name is 4-α-D-glucan glucohydrolase. The CAS number for glucoamylase is 9032-08-0.
Genencor states that T. reesei is the production organism and donor organism for their glucoamylase. Genencor states that T. reesei is a non-toxigenic and non-pathogenic fungus and has a long history of safe use in industrial scale enzyme production. Genencor discusses a literature search that they conducted for the organism and states that they found no reports implicating T. reesei in disease, toxicity, or allergenicity in healthy adults. T. reesei is classified as a Biosafety Level 1 (BSL1) microorganism by the ATCC. BSL1 microorganisms are not known to cause diseases in healthy humans. Genencor states that T. reesei is also known as Hypocrea jecorina.
According to Genencor, the production strain T. reesei 70H2-TrGA #32-9 was derived from T. reesei strain RL-P37 using recombinant DNA methods. 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 to produce cellulases for over fifteen years. Genencor also notes that FDA issued “no questions” letters in response to GRAS Notices GRN000230 (chymosin enzyme preparation), GRN000315 (transglucosidase enzyme preparation), and GRN000333 (acid fungal protease enzyme preparation), which discussed enzyme preparations produced by derivatives of this strain lineage.
Genencor states that the glucoamylase production strain was constructed using two glucoamylase expression cassettes. The first cassette contained the T. reesei cbh1 promoter and terminator, genomic DNA encoding the T. reesei glucoamylase (gla1), and the Aspergillus nidulans amdS gene encoding acetamidase, which was used as a selectable marker for the transformation of the T. reesei host strain. The second cassette consisted of the T. reesei cbh1 promoter and terminator, gla1, and the T. reesei als gene encoding acetolactate synthase which was used as a selectable marker for the retransformation of one of the primary T. reesei transformants confirmed to express the first glucoamylase expression cassette. Genencor states that the final production strain was assessed by Southern blot analysis to confirm that it contains multiple copies of the glucoamylase gene integrated into chromosomal DNA and does not contain vector DNA sequences. Genencor also states that no antibiotic resistance genes were used in the construction of the production microorganism. In addition, Genencor verified the absence of the production microorganism in the final enzyme preparation, and established a specification for the commercial product.
According to Genencor, glucoamylase is manufactured by submerged pure culture fermentation of the T. reesei production strain. The production process is consistent with current good manufacturing practice (cGMP) as set forth in 21 CFR Part 110. Each production batch is initiated from a new lyophilized stock culture, which is controlled for identity, absence of foreign microorganisms, and enzyme generating ability. Fermentation is conducted under controlled conditions and the fermentation broth is periodically tested for microbial contamination. Glucoamylase is secreted to the fermentation broth and is subsequently recovered via several purification and concentration steps. The ultrafiltered concentrate is subsequently formulated to stabilize glucoamylase and adjust its activity to a desired level. The finished enzyme preparation is an aqueous solution that contains approximately 15 percent glucoamylase, 0.11 percent potassium sorbate, 0-10 percent glucose, and 0.3 percent sodium benzoate. The total organic solids (TOS) content of the glucoamylase enzyme preparation is approximately 16 percent and the average glucoamylase activity is 570 glucoamylase activity units per gram. 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 (2006).
Genencor states that the glucoamylase enzyme preparation will be used at levels from 0.2 to 0.5 kilogram (kg) per metric ton (Mt) of dry starch substance in grain processing to produce corn sweeteners. In the production of potable ethanol and brewing, the glucoamylase enzyme preparation will be used at 0.3-0.5 kg of product per Mt (kg/Mt) of dry starch substance and 2.5-5.0 kg/Mt of grist. Genencor also proposes to use glucoamylase enzyme preparation in lactic acid production at levels of 0.5-3 kg/Mt of dry substrate.
Genencor states that glucoamylase will be inactivated or removed during subsequent processing steps for all applications, and thus its carryover to the final food would be negligible. However, to provide a conservative estimate of possible human exposure, Genencor assumes that that it would be used at the highest use levels and would be carried over to the final food. Based on these assumptions, the cumulative exposure to the glucoamylase enzyme preparation from the consumption of all foods potentially containing potable alcohol and corn sweeteners would be 0.56 milligram TOS per kg body weight per day, assuming a body weight of 60 kg.
Genencor provides the results of toxicological studies for the glucoamylase enzyme preparation. The studies included: acute oral toxicity in rats; 90-day oral (gavage) study in rats; bacterial reverse mutation assay (Ames test), and in vitro mammalian chromosomal aberration test using human lymphocytes. Genencor also provided similar toxicology studies for seven other enzyme preparations derived from T. reesei, one from a traditionally modified strain and six from recombinant strains. All tested enzyme preparations were found to be non-toxic, non-mutagenic, and not clastogenic. Based on the results of these tests, Genencor concludes that the glucoamylase enzyme preparation derived from strain 70H2-TrGA#32-9 is safe for its intended uses in food processing.
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. Consistent with the recommendations of the Codex Alimentarius Commission (2003), Genencor reports conducting an amino acid sequence homology search for glucoamylase from T. reesei with known allergens listed in the Structural Database of Allergenic Proteins (SDAP, 2003). Genencor identified homology of 35 percent or greater to two non-food allergens; namely, Pen ch 13 (fungi, Ascomycota Eurotiales; Penicillium citrinum, alkaline serine protease) and Lo1 p 5 (Poales; Lolium perenne, rye grass) when using an 80-amino acid sliding window. A second search for exact match with short contiguous sequence stretches (≥ 8 amino acids) to known food allergens resulted in zero matches. Genencor concludes that based on these results and the negligible carryover of glucoamylase to food, the enzyme is unlikely to pose a risk as a food allergen.
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 glucoamylase enzyme preparation is GRAS for the intended uses, FDA did not consider whether section 301(ll) or any of its exemptions apply to foods containing the glucoamylase enzyme preparation. Accordingly, this response should not be construed to be a statement that foods that contain the glucoamylase 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 glucoamylase 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 glucoamylase 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 000372, 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 at www.fda.gov/grasnoticeinventory.
Mitchell A. Cheeseman, Ph.D.
Office of Food Additive Safety
Center for Food Safety and Applied Nutrition