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See also Generally Recognized as Safe (GRAS).
CFSAN/Office of Food Additive Safety
September 30, 2015
Dr. Vincent Sewalt
Danisco US Inc.
(operating as DuPont Industrial Biosciences)
925 Page Mill Road
Palo Alto, CA 94304
Re: GRAS Notice No. GRN 000584
Dear Dr. Sewalt:
The Food and Drug Administration (FDA) is responding to the notice, dated May 27, 2015, 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 June 1, 2015, filed it on July 2, 2015, and designated it as GRAS Notice No. GRN 000584.
The subject of the notice is cellulase enzyme preparation from Penicillium funiculosum (cellulase enzyme preparation). The notice informs FDA of the view of Danisco US Inc., operating as DuPont Industrial Biosciences (DuPont) that cellulase enzyme preparation is GRAS, through scientific procedures, for use as an enzyme in brewing, baking, and potable alcohol production at levels up to 28.3 milligrams total organic solids per kilogram (mg TOS/kg) raw materials.
Commercial enzyme preparations that are used in food processing typically contain an enzyme component that catalyzes the chemical reaction as well as substances used as stabilizers, preservatives, or diluents. Enzyme preparations may also contain components derived from the production organism and from the manufacturing process, e.g., constituents of the fermentation media or the residues of processing aids. DuPont’s notice provides information about the components in the cellulase enzyme preparation.
According to the classification system of enzymes established by the International Union of Biochemistry and Molecular Biology, cellulase is identified by the Enzyme Commission Number 188.8.131.52. Cellulase catalyzes the endohydrolysis of 1,4-}-D-glucosidic linkages in cellulose, lichenin, and cereal β-D-glucans; it will also hydrolyze 1,4-linkages in β-D-glucans also containing 1,3-linkages. The accepted name for the enzyme is cellulase. The systematic name for this enzyme is 4-(1, 3;1,4)-β-D-glucan 4-glucanohydrolase. Other names include endo-1,4-β-D-glucanase; β-1,4-glucanase; β-1,4-endoglucan hydrolase; cellulase A; cellulosin AP; endoglucanase D; alkali cellulase; cellulase A 3; celludextrinase; 9.5 cellulase; avicelase; pancellase SS; 1,4-(1,3;1,4)-β-D-glucan 4-glucanohydrolase. The CAS Registry Number for cellulase is 9012-54-8. DuPont states that the primary sequence of cellulase consists of 389 amino acids.
DuPont states that the production strain, P. funiculosum SD101, is obtained from the wild-type P. funiculosum IMI 134755 by classical mutagenesis methods. DuPont describes P. funiculosum as a Biosafety Level 1 microorganism that is a non-pathogenic and non-toxigenic source, with a history of safe use in the production of industrial enzymes. DuPont confirms that the production strain remains stable for a dozen generations, does not produce any antibiotics, and does not contain any antibiotic resistance genes.
DuPont states that the cellulase enzyme is produced by submerged fermentation of a selected pure culture of the production strain. The fermentation is carried out under controlled conditions and the culture is periodically tested until the desired enzyme production is achieved. After fermentation, the enzyme is separated from the cell debris, and recovered by a series of filtration or centrifugation steps; this is followed by a concentration step. The resulting concentrate containing the enzyme product, free of the production strain, is formulated by the addition of sodium benzoate as a stabilizer, and standardized with sorbitol. DuPont states that the entire process is performed in accordance with current Good Manufacturing Practices using raw materials of food grade quality. DuPont also states that the final enzyme preparation contains no major food allergens from the fermentation medium.
DuPont has established food grade specifications and notes that the cellulase enzyme preparation conforms to specifications established for enzyme preparations in the Food Chemicals Codex (FCC, 9th edition, 2014), 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 (JECFA, 2006). DuPont provides analytical data from three non-consecutive manufacturing lots of cellulase enzyme concentrate to demonstrate consistency with the set specifications. DuPont confirms the absence of antibiotic resistance genes, transferable rDNA sequences, antibiotics, toxins, and the production organism in the final enzyme preparation.
DuPont proposes to use cellulase enzyme preparation to hydrolyze cellulose, lichenin and cereal β-D- glucans in brewing, baking, and potable alcohol applications. The maximum level of cellulase enzyme preparation for the proposed uses corresponds to 28.3 mg TOS/kg of final food. To estimate dietary exposure to cellulase enzyme preparation, DuPont assumes that the cellulase enzyme preparation will be used at its maximum intended use levels, and that all of the enzyme preparation will remain in the final food. Based on these assumptions, DuPont estimates a daily intake of cellulase enzyme preparation from all intended uses to be 0.131 mg TOS per kg body weight per day (mg TOS/kg bw/d). DuPont states that the reaction products resulting from cellulase enzyme activity are already part of the human diet.
DuPont summarizes corroborative toxicological studies using cellulase enzyme liquid concentrate to support the safety of the enzyme for the proposed uses. Tests conducted using bacterial cells showed that the cellulase is not mutagenic both in the presence and absence of metabolic activation. DuPont also demonstrates that the enzyme is not clastogenic based on in vitro chromosomal aberration testing. DuPont uses a 90-day oral toxicity study conducted using rats to show that the consumption of cellulase enzyme liquid concentrate does not cause any treatment-related adverse effects up to the highest dose tested, i.e., 83.7 mg TOS/kg bw/d. Based on the highest dose tested in the 90-day study, and the estimated daily intake of 0.131 mg TOS/kg bw/d from the proposed use levels of cellulase enzyme preparation, DuPont calculates a margin of safety to be approximately 600. DuPont states that based on the results of these safety studies, and other information in published literature, the cellulase enzyme preparation is considered safe for human consumption.
DuPont discusses potential food allergenicity of cellulase enzyme by conducting an amino acid sequence homology search for cellulase against known allergens stored in AllergenOnline database. Neither an amino acid overlap of >35% using a window of 80 amino acids, nor matches of contiguous stretches of eight or more amino acids, was observed. DuPont further cites the conclusions of several organizations and working groups about the low risk of allergenicity posed by enzymes due to their low use levels and extensive processing of the enzyme-containing foods during manufacturing. Based on the totality of information available, DuPont concludes that it is unlikely that oral consumption of cellulase enzyme will result in allergenic responses.
Based on the data and information summarized above, DuPont concludes that cellulase enzyme preparation is GRAS for its intended use.
Section 301(ll) of the Federal Food, Drug, and Cosmetic Act (FD&C Act)
The Food and Drug Administration Amendments Act of 2007, which was signed into law on September 27, 2007, amends the FD&C Act to, among other things, add section 301(ll). Section 301(ll) of the FD&C Act prohibits the introduction or delivery for introduction into interstate commerce of any food that contains a drug approved under section 505 of the FD&C Act, 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 DuPont’s notice that cellulase 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 cellulase enzyme preparation. Accordingly, this response should not be construed to be a statement that foods that contain cellulase enzyme preparation, if introduced or delivered for introduction into interstate commerce, would not violate section 301(ll).
Based on the information provided by DuPont, as well as the information in GRAS No. GRN 000584 and other information available to FDA, the agency has no questions at this time regarding DuPont’s conclusion that cellulase enzyme preparation from Penicillium funiculosum 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 cellulase enzyme preparation from P. funiculosum. As always, it is the continuing responsibility of DuPont 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 000584, 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.
Dennis M. Keefe, Ph.D.
Office of Food Additive Safety
Center for Food Safety and Applied Nutrition
(1) FDA notes that the cellulase enzyme preparation is a mixture of a number of cellulose-degrading enzymes including three endoglucanases, two cellobiohydrolases and a beta-glucosidase, that all catalyze the hydrolysis of cellulose.
(2) SD101, also known as PF8/403-M, is deposited in the International Mycological Institute Strain Collection as IMI 378536.
(3) IMI 134755 was purchased from International Mycological Institute.