Agency Response Letter GRAS Notice No. GRN 000402
CFSAN/Office of Food Additive Safety
November 23, 2012
Gary L. Yingling
Morgan, Lewis & Bockius LLP
1111 Pennsylvania Avenue, NW
Washington, DC 20004
Re: GRAS Notice No. GRN 000402
Dear Mr. Yingling:
The Food and Drug Administration (FDA) is responding to the notice, dated September 15, 2011, that you submitted on behalf of DSM Food Specialties (DSM) 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 September 21, 2011, filed it on September 28, 2011, and designated it as GRAS Notice No. GRN 000402.
The subject of the notice is peroxidase enzyme preparation derived from a genetically modified strain of Aspergillus niger. The notice informs FDA of the view of DSM that peroxidase enzyme preparation is GRAS, through scientific procedures, for use in the bleaching of cheese whey.1 DSM states that the typical use level of peroxidase enzyme preparation is 0.012-0.014 milligrams (mg) of total organic solids (TOS) of peroxidase enzyme preparation per milliliter (ml) of cheese whey. The peroxidase enzyme-treated cheese whey is intended to be used in dairy products, bakery products, beverages, and infant formula.
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, DSM provides information about all the components of peroxidase enzyme preparation.
The system established by the International Union of Biochemistry and Molecular Biology classifies peroxidase enzyme as Enzyme Commission number 188.8.131.52. The accepted name is peroxidase and the systematic name is phenolic donor: hydrogen peroxide oxidoreductase. The CAS Registry Number of peroxidase enzyme is 9003-99-0. DSM notes that peroxidase enzyme is a homo-dimeric protein with a primary sequence of 1014 amino acids and a calculated molecular mass of 53 kiloDaltons. The principal catalytic activity of peroxidase enzyme is the oxidation of the reaction substrate (for example, carotenoids in cheese whey) in the presence of hydrogen peroxide. Hydrogen peroxide is required as a co-factor for peroxidase enzyme activity. The products of this reaction are the oxidized substrate (bleached cheese whey) and water.
DSM states that A. niger strain ISO 528 was the host strain used to develop the production strain. The host strain is derived from A. niger strain NRRL 3122, which was purchased from the United States Department of Agriculture. The host strain was developed using several classical mutagenesis steps that inactivated genes encoding glucoamylase, amylase, and protease enzymes and increased enzyme secretion capacity. The host strain was derived from a well-characterized lineage that has been used to develop production strains for other enzymes used in food. Published literature shows that A. niger is a nonpathogenic, nontoxigenic microbe with a long history of safe use in the production of enzymes used in food.
DSM describes the development of the production strain, A. niger strain MOX-54. The host strain was transformed with two cassettes. The first cassette contains a gene that was synthesized based on the Marasmius scorodonius msp1 gene encoding peroxidase enzyme. The msp1 gene was fused to a gene encoding a truncated, nonfunctional A. niger glucoamylase enzyme. The second cassette contains the Aspergillus nidulans amdS gene which was used as a selectable marker. The amdS gene was removed during development of the production strain to create a marker-free strain. The production strain contains 24 copies of the peroxidase gene and is genetically stable. Tests confirmed that the production strain does not produce mycotoxins under the fermentation conditions used during the manufacturing process.
DSM states that peroxidase enzyme is produced by submerged fed-batch fermentation of a pure culture of the production strain. The fermentation is conducted under controlled conditions and the culture is periodically tested for microbial contamination. The fermentation process consists of three steps: pre-culture, seed fermentation, and main fermentation. During fermentation, peroxidase enzyme and glucoamylase enzyme are secreted to the fermentation broth and are subsequently purified and concentrated to obtain a liquid product. The liquid product is stabilized with glycerol and standardized to the desired peroxidase enzyme activity. The content of TOS of the final enzyme preparation is between 11 and 16%. DSM states that the manufacturing process is performed in accordance with good manufacturing practice. The raw materials used in the fermentation and purification/concentration steps meet predefined quality standards and the raw materials used in the formulation are food grade quality.
DSM states that peroxidase enzyme preparation conforms to the specifications for enzyme preparations described in the Food Chemicals Codex (7th edition), and to the current General Specifications and Considerations for Enzyme Preparations Used in Food Processing established by the FAO/WHO Joint Expert Committee on Food Additives (2006). DSM states that peroxidase enzyme preparation does not contain antibiotics or transformable recombinant DNA. DSM states that the final peroxidase enzyme preparation contains the peroxidase enzyme, the nonfunctional glucoamylase enzyme, and other enzymes that have no functional effects in the intended food applications.
DSM intends to use peroxidase enzyme preparation at levels of 0.012-0.014 mg TOS per ml cheese whey.2 The peroxidase enzyme will be inactive in the final foods because hydrogen peroxide, which is essential to peroxidase enzyme activity, will be totally consumed during the enzymatic reaction. Furthermore, the peroxidase enzyme will be inactivated by heat treatment during cheese whey processing. DSM assumes that 100% of the enzyme TOS used in cheese whey will remain in the final foods to estimate the maximum intake of peroxidase enzyme preparation. DSM uses consumption data based on USDA’s 1994 Continuing Survey of Food Intakes by Individuals and the 1995 Diet and Health Knowledge Survey. DSM estimates the maximum intake of peroxidase enzyme preparation from the intended uses of peroxidase enzyme-treated cheese whey in dairy products, bakery products, and beverages to be 0.15 mg TOS per kilogram (kg) body weight (bw) per day (d). DSM estimates the maximum daily intake of peroxidase enzyme preparation from the intended use of peroxidase enzyme-treated cheese whey in infant formula to be 0.16 mg TOS/kg bw/d.
DSM discusses published information supporting the nontoxic nature of enzyme preparations derived from A. niger and cites published information showing that peroxidase enzymes are natural constituents in many foods. To further support the safety of peroxidase enzyme preparation, DSM summarizes unpublished toxicological studies conducted with non-formulated peroxidase enzyme concentrate. DSM states that peroxidase enzyme concentrate was produced using the same procedure as the commercial peroxidase enzyme preparation. A 90-day oral toxicity study conducted using rats showed that consumption of peroxidase enzyme concentrate did not cause adverse effects at the highest dose tested (35 mg TOS per gram of feed) leading to an overall no observed adverse effect level of 2.3 grams TOS/kg bw/d. The results of chromosomal aberration tests showed that peroxidase enzyme concentrate is not clastogenic to cultured human lymphocytes. Tests conducted using bacterial cells showed that peroxidase enzyme concentrate is not mutagenic. Based on these results, DSM concludes that peroxidase enzyme preparation does not cause toxicity following oral exposure nor is it genotoxic.
DSM discusses potential allergenicity of peroxidase enzyme. DSM states that enzymes are unlikely to be food allergens, that ingestion of enzymes is typically very low, and that residual enzymes in final foods are digested in the gastrointestinal system. DSM conducted amino acid sequence identity comparisons between their peroxidase enzyme and glucoamylase enzyme and known allergens included in publicly available allergen databases (Allermatch and the Structural Database of Allergenic Proteins). The results of these analyses revealed no meaningful sequence identity (35% or more) between the peroxidase enzyme sequence, glucoamylase enzyme sequence, and the amino acid sequences of known allergens. Based on these results, DSM concludes that oral consumption of peroxidase enzyme is not likely to produce any allergenic reactions.
Based on the data and information summarized above, as well as the publicly available literature, DSM concludes that peroxidase enzyme preparation is GRAS for the intended uses.
Intended Use in Infant Formula
Under section 412 of the Federal Food, Drug, and Cosmetic Act (FD&C Act), a manufacturer of a new infant formula must make a submission to FDA, providing required assurances about the formula, at least 90 days before the formula is marketed. DSM should be aware that FDA’s response to DSM’s GRAS notice does not alleviate the responsibility of any infant formula manufacturer who intends to market an infant formula that contains peroxidase enzyme-treated cheese whey to make the submission required by section 412.
Section 301(ll) of the 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 DSM’s notice that peroxidase 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 peroxidase enzyme preparation. Accordingly, this response should not be construed to be a statement that foods that contain peroxidase enzyme preparation, if introduced or delivered for introduction into interstate commerce, would not violate section 301(ll).
Based on the information provided by DSM, as well as other information available to FDA, the agency has no questions at this time regarding DSM’s conclusion that peroxidase 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 peroxidase enzyme preparation. As always, it is the continuing responsibility of DSM 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 000402, 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
1In their original notice, DSM stated their intent to use peroxidase enzyme preparation in the bleaching of cheese whey, soy milk, and cream. In a letter dated April 20, 2012, DSM withdrew the intended uses in soy milk and cream. Thus, our evaluation of GRN 000402 was limited to the intended use of peroxidase enzyme preparation in the bleaching of cheese whey.
2In the notice, DSM expresses the use levels of peroxidase enzyme preparation in dairy bleaching units (DBLU) per milliliter of cheese whey. 1 DBLU/ml cheese whey corresponds to 0.012-0.014 mg TOS per ml cheese whey.