Agency Response Letter GRAS Notice No. 000528

Return to inventory listing: GRAS Notice Inventory

See also Generally Recognized as Safe (GRAS) and about the GRAS Notice Inventory

CFSAN/Office of Food Additive Safety

December 23, 2014

Alexander Sulakvelidze, Ph.D.
Intralytix, Inc.
701 E. Pratt Street
Baltimore, MD 21202

Re: GRAS Notice No. GRN 000528

Dear Dr. Sulakvelidze:

The Food and Drug Administration (FDA) is responding to the notice, dated July 14, 2014, 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 July 15, 2014, filed it on July 31, 2014, and designated it as GRAS Notice No. GRN 000528.

The subject of the notice is a preparation containing six bacterial monophages specific to Listeria monocytogenes (LM phage preparation). The notice informs FDA of the view of Intralytix, Inc. (Intralytix) that LM phage preparation is GRAS, through scientific procedures, for use as an antimicrobial agent in fish and shellfish (including smoked fish), fresh and processed fruits and vegetables, and dairy products including cheese at levels of up to 108 plaque forming units per gram (PFU/g) of food. FDA notes that the subject of GRN 000528 is the same preparation specified in 21 CFR 172.785 “Listeria-specific bacteriophage preparation” for use in ready-to-eat meats.

Our use of “LM phage preparation” 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.

Intralytix describes the identity and composition of LM phage preparation, which consists of a fluid suspension of six double-stranded DNA lytic phages specific to L. monocytogenes present in equal amounts at a mean total lytic titer of 10 ± 0.33 log PFU per milliliter (mL). Intralytix reports that five of the phages are classified under the order Caudovirales, family Myoviridae, and are designated LMSP-25, LMTA-34, LMTA-57, LMTA-94, and LMTA-148. The other phage is classified under order Caudovirales, family Siphoviridae and is designated as LIST-36. Intralytix provides information on the genome of the individual phages, including size, percent guanine and cytosine, and numbers of open reading frames (verifying the number of transcribed genes). Intralytix states that for application, the preparation is diluted ten-fold in water, yielding a working solution with a lytic titer of 9.0 ± 0.33 log PFU/mL. The application process ensures that the food receives not more than 108 PFU/g.

Intralytix describes the method of manufacture for LM phage preparation. Individual phages are prepared through aerobic fermentation of a suitable host L. monocytogenes strain in a soy and yeast-derived peptone containing medium to an appropriate cell density. The culture is then infected with the specified phage at an appropriate multiplicity of infection (ratio of phage to bacteria) for phage production, and then incubated under aeration with mixing. The suspension is filtered, concentrated, washed with sodium chloride (NaCl) solution, and sterilized using filtration to 0.22 micrometers (µm). The phages are combined and diluted with NaCl (0.1 Molar) as necessary to produce a final suspension containing equal titers of each phage. Intralytix uses restriction fragment length polymorphisms to verify the identities of the component phages. Intralytix estimates the shelf life of LM phage preparation at one year under dark, ultraviolet-light protected storage conditions at 2–6°C.

Intralytix provides specifications for LM phage preparation including lytic titer, bacterial sterility, and limits on the level of Listeriolysin O (LLO) (none detected at a detection limit of 5 Hemolytic Units per mL).(1) Intralytix also provides the results of lot analyses and typical composition of LM phage preparation. Typical values are provided for total nitrogen (1.8 milligrams per liter (mg/L)) and total organic carbon (17.7 mg/L).

Intralytix estimates the dietary exposure to LM phage preparation. Using food availability (per capita) data for 2012 from the U.S. Department of Agriculture, Economic Research Service, Intralytix estimates 1575 g of target foods consumed per person per day, based on per capita intakes of all fish and shellfish, all fruits and vegetables (processed and unprocessed), and all dairy (including cheese, fluid milk, butter, frozen products, and dairy powders). In its calculation of dietary exposure, Intralytix assumes 100% market saturation of the intended use of LM phage preparation in the notified food categories, yielding a total exposure of 1.6 x 1011 PFU per person per day (PFU/p/d) or 46.2 micrograms of phages/p/d (based on an estimated mass of 2.94 x 10-16 g for one phage). Intralytix noted that if LLO were present at the limit of detection, the intended uses of LM phage preparation would be safe.

Intralytix discusses the safety of phages in general as well as the safety of the LM phage preparation itself. Intralytix notes that the scientific literature extensively documents that lytic phages, which lyse host bacteria without their nucleic acids integrating into the host genome, pose no safety hazards to humans. Phages are ubiquitous in the environment, food, and in human and animal guts and oral cavities. Because they are present on all fresh, unprocessed food, and in drinking water, they are consumed on a daily basis. Humans consume and shed many phages into the environment each day. Intralytix considers the safe therapeutic use of phages for over 80 years and their specificity to particular bacterial species and strains to support their safety. Intralytix considers that phages would not have deleterious effects on the human microbiota because of this specificity.

Intralytix states that based on DNA sequencing and bioinformatics comparisons of the individual phage genome sequences to known sequences, the phages are free of undesirable genes, including genes encoding bacterial toxins and antibiotic resistance genes, and 16S RNA sequences.(2) Further, the estimated exposure to phages in the LM phage preparation by the consumer is low. Intralytix notes that the particular phages were obtained from the environment, where phages are ubiquitous. Intralytix cites a publication in which LM phage preparation had been administered (90 days) via gavage to C57BL/6J mice without notable impact on microflora and without side effects. Based on publically available information, Intralytix concludes LM phage preparation is GRAS for its intended use.

Standards of Identity

In the notice, Intralytix states its intention to use LM phage preparation in several food categories, including foods for which standards of identity exist, located in Title 21 of the Code of Federal Regulations. We note that an ingredient that is lawfully added to food products may be used in a standardized food only if it is permitted by the applicable standard of identity.

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 Intralytix’s notice that LM phage preparation is GRAS for the intended uses, FDA did not consider whether section 301(ll) or any of its exemptions apply to foods containing LM phage preparation. Accordingly, this response should not be construed to be a statement that foods that contain LM phage preparation, if introduced or delivered for introduction into interstate commerce, would not violate section 301(ll).


Based on the information provided by Intralytix, as well as other information available to FDA, the agency has no questions at this time regarding Intralytix’s conclusion that LM phage 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 LM phage preparation. As always, it is the continuing responsibility of Intralytix 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 000528, 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


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

(1)Intralytix states that LLO, a toxin produced by L. monocytogenes, was not detected in any lots. FDA considered the safety concerns of LLO during the review of Intralytix’s food additive petition (FAP 2A4738) and found that if LLO was present, it would not present a toxicological concern at the limit of detection (71 FR 47729).

(2)Intralytix compared a complete phage sequence to all sequences contained in GenBank and other databases available through the National Center for Biotechnology Information.

Page Last Updated: 02/04/2015
Note: If you need help accessing information in different file formats, see Instructions for Downloading Viewers and Players.
Language Assistance Available: Español | 繁體中文 | Tiếng Việt | 한국어 | Tagalog | Русский | العربية | Kreyòl Ayisyen | Français | Polski | Português | Italiano | Deutsch | 日本語 | فارسی | English