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  1. GRAS Notice Inventory

Agency Response Letter GRAS Notice No. GRN 000594

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See also Generally Recognized as Safe (GRAS).

CFSAN/Office of Food Additive Safety

December 30, 2015

Dr. Vincent Sewalt, Ph. D. DuPont Industrial Biosciences
925 Page Mill Road
Palo Alto, CA 94304

Re: GRAS Notice No. GRN 000594

Dear Dr. Sewalt:

The Food and Drug Administration (FDA) is responding to the notice, dated July 31, 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 August 3, 2015, filed it on August 27, 2015, and designated it as GRAS Notice No. GRN 000594.

The subject of the notice is alpha-amylase enzyme preparation produced by Bacillus licheniformis carrying an alpha-amylase gene from Geobacillus stearothermophilus. The notice informs FDA of the view of DuPont Industrial Biosciences (DuPont) that the alpha-amylase enzyme preparation is GRAS, through scientific procedures, for use as an enzyme in starch processing from grains and tubers, in brewing and production of cereal beverage, and in the production of potable alcohol, at levels of up to 31.6 milligrams total organic solids per kilogram (TOS/kg) of raw material used.

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 alpha-amylase enzyme preparation.

According to the classification system of enzymes established by the International Union of Biochemistry and Molecular Biology, alpha-amylase is identified by the Enzyme Commission Number 3.2.1.1. Alpha-amylase catalyzes the endohydrolysis of (1→4)-α-D-glucosidic linkages in polysaccharides containing threeormore(1→4)-α-linked D-glucose units. The systematic name for this enzyme is 4-α-D-glucan glucanohydrolase. Other names include glycogenase;a amylase, a-amylase; endoamylase; taka-amylase A; 1,4-α-D-glucan glucanohydrolase. The CAS Registry Number for beta-glucanase is 9000-90-2. DuPont states that the primary sequence of alpha-amylase consists of 456 amino acids.

DuPont states that the production strain is obtained from B. licheniformis Bra 7, an industrial strain developed from its wild-type parent via classical strain improvement techniques. DuPont describes B. licheniformis as a common bacterium found in the soil. It is a non-pathogenic and non-toxigenic source with a history of safe use in the production of industrial enzymes. DuPont states that B. licheniformis Bra7 has been used as a host for production of several enzymes added to food including glycerophospholipid cholesterol acyltransferase (GRAS No. GRN 265) and maltotetraohydrolase (GRAS No. GRN 277).

DuPont describes the construction of the production strain. The host B. licheniformis BRA7 strain is modified by classical strain improvements to decrease protease production, increase D-amylase activity, and to remove sporulation capability and native chloramphenicol acyltransferase activity. The improved strain was subsequently transformed with a plasmid vector carrying the expression cassette with the wild type truncated G. stearothermophilus amyS gene encoding alpha-amylase under the control of a B. licheniformis amyL promoter and terminator, and the native B. licheniformis cat gene. After integration, all vector sequences of the plasmid were deleted, leaving only the truncated G. stearothermophilus amyS gene and the native cat gene. DuPont verified the final genetic construct by Southern blot analysis. DuPont confirms that the production strain is stable after industrial scale fermentation, and that it does not produce any antibiotics, or contain any antibiotic resistance genes.

DuPont states that the alpha-amylase enzyme is produced by submerged fermentation of a 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 recovered from the culture broth by a primary separation step followed by a concentration step. The resulting enzyme concentrate is formulated by the addition of sorbitol, sodium chloride, propylene glycol, calcium chloride, potassium sorbate, and paraben, at pH 5.0-6.7. 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 alpha-amylase enzyme preparation contains no major food allergens from the fermentation medium.

DuPont has established food grade specifications and notes that the alpha-amylase enzyme preparation conforms to specifications established for enzyme preparations in the Food Chemicals Codex (FCC, 9th edition, 2014) and 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 confirms the absence of the production organism in the final enzyme preparation, as established by the set specifications. DuPont also provides analytical data from three batches of alpha-amylase enzyme produced to demonstrate consistency with the manufacturing specifications.

DuPont intends to use alpha-amylase enzyme preparation in the processing of starch, brewing of cereal beverages, and production of potable alcohol, to hydrolyze (1-4)-D-D-glucosidic linkage in polysaccharides containing three or more (1-4)-D-linked-D-glucose units. The maximum intended use level of alpha-amylase enzyme preparation is 31.6 mg TOS/kg of raw material. DuPont estimates the dietary exposure to alpha-amylase enzyme preparation from all intended uses to be 0.14 mg TOS per kg body weight per day (mg TOS/kg bw/d). DuPont assumes that all of the enzyme preparation will remain in the final food, in this estimation. DuPont also states that the reaction products resulting from alpha- amylase enzyme activity are already part of the human diet.

DuPont summarizes toxicological studies based on safe strain lineage of the alpha-amylase enzyme. DuPont demonstrates safety of the alpha-amylase enzyme preparation based on in vitro and in vivo studies conducted with another alpha-amylase that is also produced by B. licheniformis, and carries an alpha-amylase gene from G. stearothermophilus. DuPont also summarizes reviews of safety studies of enzyme preparations from different strains of B. licheniformis to demonstrate that the subject of this notice is not mutagenic, clastogenic or aneugenic. DuPont states that the alpha-amylase enzyme will be inactivated during processing, and any inactive enzyme, if consumed, would be absorbed and metabolized via well-documented and scientifically accepted biochemical pathways, which are considered general scientific principles.

DuPont examines the potential allergenicity of alpha-amylase enzyme by conducting an amino acid sequence homology search for alpha-amylase against known allergens stored in Food Allergy Research and Resource Program’s FARRP AllergenOnline database for sequence identity matches over 35%. DuPont reported search results that revealed multiple stretches throughout the peptide sequence with 38% identity to TAKA-amylase-A, an environmental and occupational allergen, and also referred to as Asp o 21. DuPont states that TAKA-amylase-A has not been identified as a food allergen by the World Health organization and International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Sub-committee. Also, DuPont did not find any matches of contiguous stretches of eight or more amino acids in the alpha-amylase sequence that would be cross reactive with an allergen protein. 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 alpha-amylase enzyme will result in allergenic responses.

Based on the data and information summarized above, DuPont concludes that alpha-amylase enzyme preparation is GRAS for its intended use.

Section 301(ll) of the Federal Food, Drug, and Cosmetic Act (FD&C Act)

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 alpha-amylase 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 alpha-amylase enzyme preparation. Accordingly, this response should not be construed to be a statement that foods that contain alpha-amylase enzyme preparation, if introduced or delivered for introduction into interstate commerce, would not violate section 301(ll).

Conclusions

Based on the information provided by DuPont, as well as other information available to FDA, the agency has no questions at this time regarding DuPont’s conclusion that alpha-amylase 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 alpha-amylase enzyme preparation.  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 000594, 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.

Sincerely,

Dennis M. Keefe, Ph.D. Director
Office of Food Additive Safety
Center for Food Safety and Applied Nutrition

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