Agency Response Letter GRAS Notice No. GRN 000333
CFSAN/Office of Food Additive Safety
September 24, 2010
Alice J. Caddow
Genencor, a Danisco Division
925 Page Mill Road
Palo Alto, CA 94304
Re: GRAS Notice No. GRN 000333
Dear Ms. Caddow:
The Food and Drug Administration (FDA) is responding to the notice, dated March 29, 2010, 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 March 30, 2010, filed it on March 31, 2010, and designated it as GRAS Notice No. GRN 000333.
The subject of the notice is acid fungal protease (AFP) enzyme preparation from Trichoderma reesei expressing the gene encoding acid fungal protease from T. reesei (AFP enzyme preparation). The notice informs FDA of the view of Genencor, a Danisco Division (Genencor) that AFP enzyme preparation is GRAS, through scientific procedures, for use as an enzyme in grain processing (corn steeping), alcoholic beverage (potable alcohol) manufacture, and degumming of membranes during orange juice manufacturing.
As part of its notice, Genencor includes the report of a panel of individuals (Genencor’s GRAS panel) who evaluated the data and information that are the bases for Genencor’s GRAS determination. Genencor considers the members of its GRAS panel to be qualified by scientific training and experience to evaluate the safety of substances added to food. Genencor’s GRAS panel evaluated: 1) development of the production organism and safety of the host strain; 2) method of manufacture, product composition, and specifications; 3) intended uses and estimates of dietary exposure; and 4) toxicological studies. The panel stated that there are no reports of disease, toxicity, or allergenicity associated with T. reesei. Based on its review, Genencor’s GRAS panel concluded that the AFP enzyme preparation is GRAS under the intended conditions of use.
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 manufacturing process. In their notice, Genencor provides information about each of these components of the AFP enzyme preparation.
Genencor provides specific information about the identity and activity of AFP. According to the classification system of enzymes established by the International Union of Biochemistry and Molecular Biology (IUBMB), AFP is identified by the Enzyme Commission number 188.8.131.52. Its accepted name is aspergillopepsin I. The Chemical Abstract Service Registry number for AFP is 9025-49-4. AFP is a protease with broad specificity, which primarily catalyzes the hydrolysis of proteins at hydrophobic residues.
Genencor discusses the safety of the production organism, T. reesei, which is also known in the literature as Hypocrea jecorina. Genencor states that T. reesei is nonpathogenic and nontoxigenic based on published literature and its history of use in enzyme production. T. reesei has been classified as a Biosafety Level 1 (BSL1) microorganism by the American Type Culture Collection. BSL1 microorganisms are not known to cause disease in healthy humans.
Genencor describes the development of the T. reesei production strain designated NSP24 #22-1. Genencor states that strain NSP24 #22-1 was derived from the host strain RL-P37 by recombinant DNA methods. The RL-P37 strain was developed from the well-known wild-type strain QM6a using classical mutagenesis. Genencor reports that the strain has been determined to be nonpathogenic and nontoxicogenic through intraperitoneal dosing of rats. The T. reesei strain RL-P37 has been used by Genencor for over fifteen years for the production of cellulases. Genencor also notes that FDA issued a ‘no questions’ letter in response to GRN 000230, the subject of which is bovine chymosin obtained from a T. reesei production strain that was derived from strain RL-P37.
Genencor states that the host strain RL-P37 was transformed with an expression vector containing the DNA encoding the T. reesei mature secreted AFP protein fused to the DNA encoding the T. reesei CBH1 signal peptide. Genencor states that the open reading frame is flanked by the promoter and terminator sequences of the T. reesei cbh1 gene. The expression vector also contains the selectable marker gene amdS from Aspergillus nidulans. Genencor states that the incorporation of the desired genetic information was evaluated at every step of the construction. The final construct was verified by Southern blot analysis to confirm that only the intended genetic modifications to the T. reesei production strain had been made. Genencor states that the production strain is genetically stable, and that no antibiotic resistance genes were used in the development of the production strain.
Genencor states that AFP is produced during cultivation of the production strain using standard fermentation procedures. The fermentation is conducted under controlled conditions and is periodically monitored for microbial contamination. AFP is secreted to the fermentation broth and is subsequently recovered via several purification and concentration steps. The resulting liquid concentrate (AFP concentrate) is stabilized with food-grade glycerol, sodium sulfate, and sodium benzoate, and is tested to demonstrate it meets product specifications. The final enzyme preparation contains 2.8-3% AFP and 5.1-5.5% total organic solids (TOS). Genencor states that the manufacturing process is performed in accordance with current Good Manufacturing Practice (cGMP) as set forth in 21 CFR Part 110. Genencor states that the AFP enzyme preparation conforms to the specifications for enzyme preparations described in the Food Chemicals Codex (6th edition) and to 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 AFP enzyme preparation will be used in grain processing at 9 micrograms (μg) per gram (g), in alcoholic beverage manufacturing at 160 ug per milliliter, and in degumming equipment membranes in orange juice manufacturing. To estimate human exposure to the AFP enzyme preparation, Genencor assumes that AFP enzyme preparation would be used at the highest intended use levels for grain processing and alcoholic beverage manufacturing and that 100% of the enzyme would be carried over to the final food product. In actuality the carryover of AFP enzyme preparation will be negligible. Genencor states that AFP used in degumming filter membranes during orange juice processing is not expected to be carried over to the juice because the membranes are extensively washed after degumming prior to re-use in the juice production. Given these assumptions, the cumulative exposure to AFP enzyme preparation would be 147 μg of TOS per kilogram bodyweight per day (μg TOS/kg bw/d)
Genencor summarizes toxicological studies conducted with the AFP concentrate containing 14.3 percent TOS. The studies include acute (14-day) and sub-chronic (91-day) oral toxicity tests in rats, a bacterial reverse mutation assay (Ames test), and two chromosomal aberration tests using human lymphocytes. Genencor concludes that the AFP concentrate was not toxic in the acute toxicity study and that no treatment-related effects were observed in the subchronic study at the highest dose tested of 35.8 mg TOS/kg bw/d. Genencor also states that the AFP concentrate did not induce chromosomal aberrations in human lymphocytes and has not shown mutagenic activity in the Ames test.
Genencor provides information regarding the potential allergenicity of AFP and of enzymes used in food processing in general. Consistent with the recommendations of the Codex Alimentarius Commission (2003), Genencor performed an amino acid sequence homology search for AFP against known allergens listed in the publicly available Structural Database of Allergenic Proteins (SDAP). Genencor identified a significant homology (> 35% identity within multiple 80 amino acid windows) with the respiratory allergen aspergillopepsin (Asp f 10) from Aspergillus fumigatus. The notifier subsequently analyzed the hydrophilicity of AFP and concluded that none of the matching sequences is hydrophilic and therefore would not be exposed on the surface of a folded protein where it could act as an immunogenic epitope. Genencor also notes that Asp f 10 is not a recognized food allergen and that environmental allergens are generally not known to be active via oral exposure. Further, Genencor states that the actual carry-over of AFP into products from grain processing would be negligible and insufficient to invoke an allergic reaction even if the AFP protein were a food allergen.
Section 301(ll) of the Federal Food, Drug, and Cosmetic Act (FFDCA)
The Food and Drug Administration Amendments Act of 2007, which was signed into law on September 27, 2007, amends the FFDCA to, among other things, add section 301(ll). 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 AFP enzyme preparation is GRAS for the intended use, FDA did not consider whether section 301(ll) or any of its exemptions apply to foods containing AFP enzyme preparation. Accordingly, this response should not be construed to be a statement that foods that contain the AFP 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 AFP 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 AFP 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 000333, 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