Agency Response Letter GRAS Notice No. GRN 000593
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See also Generally Recognized as Safe (GRAS).
CFSAN/Office of Food Additive Safety
December 18, 2015
Kristi Smedley, PhD
Center for Regulatory Services, Inc.
5200 Wolf Run Shoals Road
Woodbridge, VA 22192
Re: GRAS Notice No. GRN 000593
Dear Dr. Smedley:
The Food and Drug Administration (FDA) is responding to the notice, dated July 24, 2015, that you submitted on behalf of Nomad Bioscience GmbH (Nomad) 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 July 28, 2015, filed it on August 6, 2015, and designated it as GRAS Notice No. GRN 000593.
The subject of the notice is antimicrobial preparations containing one or more colicin proteins (colicin preparations). The notice informs FDA of the view of Nomad that colicin preparations are GRAS, through scientific procedures, for use as antimicrobial treatments on fresh or processed fruits1 and vegetables, at a level of 1-10 milligrams (mg) of colicin preparations per kilogram (kg) of fruit or vegetable product.
Our use of “colicin preparations” in this letter should not be considered an endorsement or recommendation of that term as an appropriate common or usual name for the purpose of declaring the substance in the ingredient statement of foods that contain that ingredient. Title 21 CFR 101.4 states that each ingredient must be declared by its common or usual name. In addition, 21 CFR 102.5 outlines general principles to use when establishing common or usual names for nonstandardized foods. Issues associated with labeling and the appropriate common or usual name of a food are the responsibility of the Office of Nutrition, Labeling, and Dietary Supplements in the Center for Food Safety and Applied Nutrition.
Nomad describes nine recombinant colicin proteins intended for use singly or in combination in colicin preparations: colicin E1, colicin E7, colicin Ia, colicin M, colicin N, colicin K, colicin U, colicin 5, and colicin B. These proteins belong to the colicin family of bacteriocins and are synthesized by Escherichia coli or by other enteric bacteria in the human intestine. Nomad states that the recombinant colicins are identical to the colicins produced by enteric bacteria and provides the CAS number (if applicable), amino acid sequence, UniProt protein sequence database entry, and molecular weight of each colicin (Table 1).
|Colicin||CAS Number||UniPort Entry||Molecular Weight (kDa)|
Nomad describes the method of manufacture of colicin preparations. Each recombinant colicin protein is produced in leaves of spinach (Spinacia oleracea), red beet (Beta vulgaris), or lettuce (Lactuca sativa). All plants are grown indoors under environmentally controlled conditions under principles of good agriculture and collection practices.2 Each protein is expressed from recombinant Tobacco mosaic virus or Potato virus X engineered to contain a colicin gene. The plant viral vector replicates in the host plant resulting in accumulation of colicin protein. These viruses are nonpathogenic to animals and are inactivated in subsequent protein isolation steps. The viral vector may be introduced to the plant either by Agrobacterium tumefaciens-mediated transient expression or by ethanol induction of stably transformed plants. After induction by either method, the plants are incubated for five to ten days to allow for colicin accumulation, and leaves and stems are homogenized. After removing insoluble material, protein is enriched by a series of acid precipitation, centrifugation, and filtration steps. Nomad intends to offer colicin preparations with two different degrees of purity: lower purity “colicin concentrate” estimated to be 30 to 60 percent pure and higher purity “colicin isolate” estimated to be 80 to 90 percent pure. The more purified preparation undergoes an additional ion-exchange chromatography step. Nomad notes that residual materials derive from safely consumed plants and are not expected to raise safety issues. Nomad states that it manufactures colicin preparations according to current good manufacturing practices. All raw materials and processing aids are food grade.
Nomad provides food grade specifications for colicin isolate and colicin concentrate preparations. Specifications include limits for total heavy metals (<30 mg/kg), lead (<5 mg/kg), microbial contaminants (including Agrobacterium), and acceptance criteria for specific activity, physical properties, and stability (>6 months).
Nomad estimates dietary exposure to colicins to be 4.1 mg/person/day based on the intended uses at the maximum application rate of 10 mg/kg of fruits and vegetables using data on per capita consumption from the United States Department of Agriculture Food Availability (Per Capita) Data System.
Nomad notes that humans are normally exposed to colicin-producing bacteria and to colicins. Citing published information, Nomad approximates that humans are chronically exposed to 3 mg colicins per day from colicin-producing bacteria in the colon. Nomad discusses published studies as well as its own unpublished studies demonstrating that colicins, including the plant-produced colicins, are rapidly digested by proteases in the gastrointestinal tract.
Nomad discusses one published and one unpublished study demonstrating the safety of colicins in piglets. In the published study, piglets fed a control diet including a pathogenic E. coli strain for four days succumbed to the effects of diarrhea and had significantly reduced feed consumption and body weight gain, whereas animals fed the pathogenic E. coli strain and a diet containing up to 16.5 mg colicin E1 per kg of feed demonstrated typical body weight gain and feed consumption. In the unpublished study, piglets fed a diet containing up to 20 mg of colicin E1 per kg of feed for 4 weeks showed no significant differences in body weight compared to piglets fed a control diet.
Nomad notes that plant-produced colicins are not expected to be allergenic or immunogenic to humans because they are rapidly degraded by gastrointestinal proteases. They also lack glycosylation and have the same amino acid sequences as bacterial colicins, which are not known to be allergenic. A comparison of the amino acid sequences of colicins to a database of known allergens found low potential for allergenicity using criteria proposed in Codex Alimentarius guidelines.3 Nomad also notes that consumption of colicins at any level is unlikely to promote colicin resistance among bacteria in the human colon because colicins are rapidly inactivated by the low pH of the stomach and are digested by gastrointestinal proteases.
Some Uses May Require Regulatory Actions by the United States Environmental Protection Agency (EPA)
Antimicrobial agents used on raw agricultural commodities may require registration as pesticides with EPA under the Federal Insecticide, Fungicide, and Rodenticide Act. FDA’s evaluation of this GRAS notice does not relieve the obligation to register colicin preparations as a pesticide for uses regulated by EPA. For information about the regulatory status of your product when used as a pesticide, please contact EPA’s Office of Pesticide Products, Antimicrobial Division.
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 Nomad’s notice that colicin preparations are GRAS for the intended uses, FDA did not consider whether section 301(ll) or any of its exemptions apply to foods containing colicin preparations. Accordingly, this response should not be construed to be a statement that foods that contain colicin preparations, if introduced or delivered for introduction into interstate commerce, would not violate section 301(ll).
Based on the information provided by Nomad, as well as other information available to FDA, the agency has no questions at this time regarding Nomad’s conclusion that colicin preparations are 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 colicin preparations. As always, it is the continuing responsibility of Nomad 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 000593, 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) Intended uses in processed products include only those that are uncooked, i.e. washed, bagged, chopped, peeled, or otherwise mechanically processed.
(2) Nomad notes that plant-derived residuals are discarded.
(3) CODEX Alimentarius. 2003. Report of the Fourth Session of the Codex Ad Hoc Intergovernmental Task Force on Foods Derived from Biotechnology. CODEX Alimentarius, Yokohama, Japan.