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Agency Response Letter GRAS Notice No. GRN 000598

Recently Published GRAS Notices and FDA Letters

See also Generally Recognized as Safe (GRAS).

CFSAN/Office of Food Additive Safety

February 29, 2016

Mr. Melvin Drozen
Keller and Heckman, LLP
1001 G Street, N.W.
Suite 500 West
Washington, DC 20001

Re: GRAS Notice No. GRN 000598

Dear Mr. Drozen:

This letter corrects our letter dated September 14, 2016, sent in response to GRAS Notice No. GRN 000598. The purpose of this revised letter is to correct information regarding the scope of uses of the subject of the notice.

The Food and Drug Administration (FDA) is responding to the notice dated September 2, 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), on behalf of Amano Enzyme, Inc. (Amano). FDA received this notice on September 4, 2015, filed it on September 11, 2015, and designated it as GRAS Notice No. GRN 000598. In an amendment dated November 30, 2015, Amano modified the intended conditions of use to be those as stated in the following paragraph.

The subject of the notice is thermolysin enzyme preparation produced by Geobacillus stearothermophilus (thermolysin enzyme preparation). The notice informs FDA of the view of Amano that thermolysin enzyme preparation is GRAS, through scientific procedures, for use as an enzyme in the production of yeast extract; meat extract; egg white and yolk hydrolysates; processed egg products; enzyme-modified dairy ingredients; gelatin; cooked fish; cooked beef; cooked chicken; cooked pork; and protein hydrolysates from soy, wheat gluten, milk, gelatin, meat, chicken, and fish, to improve solubility, taste, and digestibility, at a maximum use level of 0.08% of thermolysin.

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 components derived from the manufacturing process, e.g., constituents of the fermentation media or the residues of processing aids. Amano’s notice provides information about each of these components in the thermolysin enzyme preparation.

According to the classification system of enzymes established by the International Union of Biochemistry and Molecular Biology, thermolysin is identified by the Enzyme Commission Number The accepted name for the enzyme is thermolysin; it has not been assigned a systematic name. The CAS Registry Number for thermolysin is 9073-78-3. Other names include Bacillus thermoproteolyticus neutral proteinase; thermoase; thermoase Y10; TLN. Thermolysin catalyzes the hydrolysis of peptide bonds with a preferential cleavage at leucine and phenylalanine. Amano notes that the primary amino acid sequence of thermolysin contains 316 amino acids and has been described in the literature.

Amano describes the G. stearothermophilus[1] as a non-toxigenic and non-pathogenic thermophilic, rod-shaped, Gram positive bacterium, with a history of safe use in the food industry. Amano states that the production strain was generated by exposing the wild-type strain to several rounds of classical chemical mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (NTG). The resulting modified strain (the production strain) overproduces the thermolysin enzyme. Amano verified that the nucleotide sequence of the thermolysin gene was unchanged in the production strain following treatment with NTG. Amano states that the production strain does not produce any antibiotics, or contain any antibiotic resistance genes.

Amano states that thermolysin is produced through the controlled aerobic fermentation of a pure culture of G. stearothermophilus production strain.The enzyme is recovered from the fermentation broth via multiple filtration steps followed by concentration, crystallization, and spray drying to a final powder form. Amano states that the production process includes microfiltration of particles greater than 0.2 μm to remove the production organism.[2] The concentrated spray dried enzyme is standardized by the addition of sodium chloride as a diluent and stabilizer. Amano states that the entire process is performed in accordance with current Good Manufacturing Practices using raw materials of food grade quality. Amano also states that the final thermolysin enzyme preparation contains no major food allergens from the fermentation medium.

Amano has established food grade specifications and notes that the thermolysin enzyme preparation conforms to specifications established for enzyme preparations in the Food Chemicals Codex (FCC, 9th edition, 2015) 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). Amano provides analytical data from five batches of thermolysin enzyme concentrate to demonstrate consistency with the set specifications.

Amano intends to use thermolysin enzyme preparation in the production of yeast extract; meat extract; egg white and yolk hydrolysates; processed egg products; enzyme-modified dairy ingredients; gelatin; cooked fish, cooked beef, cooked chicken, and cooked pork; and protein hydrolysates from soy, wheat gluten, milk, gelatin, meat, chicken, and fish. The maximum intended use level of thermolysin enzyme preparation is 0.08% of the enzyme. Amano estimates the dietary exposure to thermolysin enzyme preparation from all intended uses to be 2.3 milligram of thermolysin preparation per kilogram body weight per day (mg thermolysin/kg bw/d).[3] In this estimation, Amano assumes that all of the enzyme preparation will remain in the final food product. Amano also states that the reaction products resulting from thermolysin enzyme activity are already part of the human diet.

Amano summarizes published toxicological studies using thermolysin enzyme concentrate to corroborate the safety of thermolysin enzyme preparation for the intended uses. Test conducted using the mouse micronucleus assay showed that thermolysin is not mutagenic. Amano also demonstrates that the thermolysin is not clastogenic based on in vitro chromosomal aberration testing. A 90 day sub-chronic oral toxicity study conducted in rats showed that consumption of thermolysin enzyme concentrate did not cause any treatment-related adverse effects up to the highest dose tested, i.e., 390 mg thermolysin/kg bw/d. Based on the highest dose tested in the 90-day study, and the estimated daily exposure from the intended use levels of thermolysin enzyme preparation, Amano calculates a margin of safety to be approximately 170.[3]

Amano discusses potential food allergenicity of thermolysin. Amano conducted an 80-amino acid sequence homology search for thermolysin enzyme against known allergens stored in the Food Allergy Research and Resource Program, FARRP, database. Amano did not identify any significant sequence homology between thermolysin and any of the allergens in this database. Amano performed an eight amino acid exact analysis of thermolysin that resulted in one match of contiguous stretch of eight amino acid sequence from an aspartic protease inhibitor 11 (Sola t 2), from potato.[4] Amano relies on the conclusions of several organizations and working groups, and scientific literature, regarding the unreliability of short sequence homology (6-8 amino acids) in concluding that this match is a false positive result. Based on the available information, Amano concludes that oral consumption of the thermolysin enzyme preparation will not result in any allergenic responses.

Use in Products under USDA Jurisdiction

During its evaluation of GRN 000598, FDA consulted with the Risk and Innovations Management Staff (RIMS) of the Food Safety and Inspection Service (FSIS) of the United States Department of Agriculture (USDA). Under the Federal Meat Inspection Act, Poultry Products Inspection Act, and the Egg Products Inspection Act, FSIS is responsible for determining the efficacy and suitability of food ingredients in meat, poultry, and egg products as well as prescribing safe conditions of use. Suitability relates to the effectiveness of the ingredient in performing the intended purpose of use and the assurance that the conditions of use will not result in an adulterated product, or one that misleads consumers.

FSIS determined that Amano has provided sufficient data to support that thermolysin enzyme preparation produced by G. stearothermophilus is suitable for use as an enzyme in the production of yeast extract; meat extract; egg white and yolk hydrolysates; processed egg products; enzyme modified dairy ingredients; gelatin; cooked fish; cooked beef; cooked chicken; and cooked pork; and protein hydrolysates from soy, wheat gluten, milk, meat, chicken, and fish, to improve the protein solubility, taste, and digestibility, at a maximum use level of 0.08% of thermolysin.

FSIS notes that Amano Enzyme, Inc. should be aware of the following labeling requirements: whole muscle meat and poultry product should be labeled as "Enzyme modified species or kind name," for example "Enzyme modified Beef Roast," or "Enzyme modified chicken breast" with "thermolysin enzyme preparation" listed in the ingredients statement; comminuted meat and poultry would simply list "thermolysin enzyme preparation" in the ingredients statement; and egg products would be labeled as "Enzyme modified (product name)" with thermolysin enzyme preparation" in the ingredients statement.

If you have any regulatory guidance questions in response to these statements from FSIS, please contact: William Shaw, Jr., Director, Risk, Innovations, and Management Staff, Office of Policy and Program Development, Food Safety and Inspection Service, Stop Code 3782, Patriots Plaza III, 8- 163A, 1400 Independence Ave., SW., Washington, DC 20250-3700 or via email at William.Shaw@fsis.usda.gov.

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


Based on the information provided by Amano, as well as other information available to FDA, the agency has no questions at this time regarding Amano’s conclusion that thermolysin enzyme preparation produced by G. stearothermophilus 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 thermolysin enzyme preparation produced by G. stearothermophilus. As always, it is the continuing responsibility of Amano 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 000598, 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] G. stearothermophilus is a reclassification of B. stearothermophilus based on phenotype and 16S rRNA gene sequence analysis.

[2] G. stearothermophilus is 0.6-1μm in width, and 2-3.5μm in length.

[3] Amano reported doses in its feeding studies in units of mg thermolysin/kg bw/d because the specific values for the levels of ash, moisture and diluents to calculate Total Organic Solids (TOS) of thermolysin preparation in the test article used in the 90-day study were unavailable. Amano reports the estimated dietary exposure to thermolysin also in mg thermolysin/kg bw/d, instead of TOS, for accurate comparison.

[4] Potatoes have been consumed by humans for centuries and without allergenicity issues. Potatoes are not on the FDA list of Major Food Allergen Food Groups.

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