Agency Response Letter GRAS Notice No. GRN 000454

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CFSAN/Office of Food Additive Safety

September 27, 2013

Claire L. Kruger, Ph.D., D.A.B.T.
Spherix Consulting, a division of ChromaDex, Inc.
11900 Parklawn Drive, Suite 200
Rockville, MD 20852

Re: GRAS Notice No. GRN 000454

Dear Dr. Kruger:

The Food and Drug Administration (FDA) is responding to the notice, dated December 21, 2012, that you submitted on behalf of Morinaga Milk Industry Co., Ltd. (Morinaga) 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 December 28, 2012, filed it on January 22, 2013, and designated it as GRAS Notice No. GRN 000454. Amendments to GRN 000454, dated June 3, and September 9, 2013, clarified the intended use.

The subject of the notice is Bifidobacterium breve strain M-16V. The notice informs FDA of the view of Morinaga that B. breve strain M-16V is GRAS, through scientific procedures, for use as an ingredient in non-exempt powdered term infant formulas (milk- or soy-based) and exempt powdered term infant formula containing partially-hydrolyzed milk or soy proteins, at levels up to 108 colony forming units (cfu) per gram of infant formula powder. Morinaga does not intend uses to include immunocompromised infants.

As part of its notice, Morinaga includes the report of a panel of individuals (Morinaga’s GRAS panel) that evaluated the data and information that are the basis for Morinaga’s GRAS determination. Morinaga considers the members of its GRAS panel to be qualified by scientific training and experience to evaluate the safety of substances added to food. Morinaga’s GRAS panel evaluated estimates of dietary exposure, method of production, and product specifications as well as published and unpublished studies. Based on this review, Morinaga’s GRAS panel concluded that B. breve strain M-16V is GRAS under the conditions of its intended use.

Morinaga describes the isolation and characterization of B. breve strain M-16V. B. breve strain M-16V was isolated from the feces of a healthy infant in 1963 and has since been stored and maintained by the Biological Function Research Department of the Food Science and Technology Institute at the Morinaga Research and Development Center. Morinaga further states that B. breve strain M-16V has been deposited as well in the Belgian Co-ordinated Collections of Microorganisms and has been designated LMG 23729.

Morinaga discusses that bifidobacteria are naturally occurring, lactic acid-producing gut microbiota, and have been consumed extensively from fermented foods. B. breve strain M-16V, like other bifidobacteria, has been characterized in the published literature as rod-shaped, anaerobic, Gram-positive, non-motile, and non-spore forming.

The notifier discusses genomic analyses that show B. breve strain M-16V is homologous to other species of Bifidobacterium that are currently used in foods and that it is nontoxic and nonpathogenic. Morinaga states that B. breve strain M-16V genome does not contain regions homologous to sequences of known antibiotic resistance genes and does not contain plasmids, induce biogenic amine formation, produce ammonia, have azoreductase or nitroreductase activity, hemolytic or mucolytic activity, or induce platelet aggregation. Morinaga notes that bile salt hydrolase activity was observed in B. breve strain M-16V and is common to all bifidobacteria. Morinaga describes the production of B. breve strain M-16V. Morinaga states that the production meets the requirements of Hazard Analysis and Critical Control Point (HACCP) System and Guidelines for its Application adopted by the Joint FAO/WHO Food Standards Programme, Codex Alimentarius Commission.(1) Further, Morinaga states that the methodology is typical of the production of microorganisms used in food as described in an earlier GRAS notice from Morinaga (GRN 268 for B. longum strain BB536) and in the published literature. Stocks of B. breve strain M-16V are stored in a medium containing skim milk, and are controlled to ensure purity at the Morinaga Laboratory. The stocks are expanded in two phases, using sterilized medium that contains yeast extract, glucose or lactose, phosphates, acetate, amino acids, and vitamins, and, for the second phase only, additional components (citrate plus sodium hydroxide or carbonate). Morinaga performs quality control processes to ensure the identity and purity of the manufacturing culture and that finished products are free of contaminants.

Morinaga then describes the preparation of the B. breve strain M-16V culture for commercial distribution. The B. breve strain M-16V culture is concentrated, washed, reconcentrated, and resuspended in medium containing sucrose or glucose, phosphates, amino acids, vitamins and agar. The culture is then freeze dried, crushed to a powder, and mixed with a food-grade carbohydrate carrier (cornstarch) at levels up to 90% of finished product. Finally, the ingredient is sifted, packed, and stored at 10°C. Morinaga describes the ingredient as a white to slightly-brown powder and provides specifications including: limits for lead (<0.2 milligrams per kilogram (mg/kg), arsenic (<1 mg/kg), Enterobacteriaceae (negative/0.01 grams (g)), Salmonella (negative/25g), Chronobacter sakazakii (negative/25 g), and proteins from milk (casein (<0.1 mg/kg) and β-lactoglobulin (<0.1 mg/kg)).

Morinaga discusses information about the stability and viability of the B. breve strain M-16V, evaluated in accordance with published methods, in a powdered infant formula currently in development in Japan and in a powdered food for older infants (6 months and up) that is currently marketed in Japan. Briefly, a powdered infant food and a powdered infant formula, each containing 1 x 108 cfu/g B. breve strain M-16V were held at 5, 25, and 37 °C for 24 months, with viability determined at 0 and 1 month and then intermittently at 3, 6, 9, 12, 18, and 24 months. Morinaga notes that B. breve strain M-16V was stable in both products, with only minimal losses observed, when stored at 5 and 25 °C. However, viability of B. breve strain M-16V decreased in a time-dependent manner in products stored at 37 °C.

Morinaga estimates consumption of B. breve strain M-16V for term infants that consume either non-exempt term infant formula or exempt term infant formula containing hydrolyzed proteins. Morinaga cites the estimated average caloric requirements of male infants reported by the Institute of Medicine (2005): 472 kilo calories perday (kcal/d) for one-month old infants and 645 kcal/d for six-month old infants. Using these caloric intake levels, the typical caloric density of infant formula of 0.67 kcal/milliliter (mL) and assuming 14.1 g powdered infant formula is added per 100 mL reconstituted formula(2) and that the maximum level of 108 cfu B. breve strain M-16V is added per gram of powdered infant formula, Morinaga estimates that daily intake of a B. breve strain M-16V is 9.9 x 109 cfu for a one-month infant and 1.35 x 1010 cfu B. breve strain M-16V/d for a six-month infant, regardless of whether exempt or non-exempt formula is consumed.

Morinaga summarizes published preclinical and clinical toxicology studies conducted with B. breve strain M-16V. Morinaga reviews a 13-week repeated dose toxicity study in which rats that consumed B. breve strain M-16V showed no adverse treatment related effects at the highest dose tested (equivalent to 2.3 x 1011 cfu/ kg/d). Morinaga also summarized numerous published clinical studies and concluded that B. breve strain M-16V, at levels up to 1.5 X 1010 cfu/d, is well tolerated by infants.

Morinaga notes that bifidobacteria and other lactic acid-producing microorganisms have been consumed in fermented foods for decades. Morinaga cites several examples of bifidobacteria strains currently used in foods including: B. lactis Bb-12 (GRN 000049) for use in infant formula for infants 4 months and older, B. longum BB536 (GRN 000268) for use in selected foods including infant formula for infants 9 months and older, and B. animalis ssp. lactis Bf-6 for use in conventional foods (GRN 000377).

Based on the totality of the data and information described above, Morinaga concludes that B. breve strain M-16V is well tolerated and is GRAS for its intended use in food.

Potential Labeling Issues

In describing data and information on clinical studies that Morinaga relies on to conclude that B. breve strain M-16V is GRAS under the conditions of its intended use, Morinaga raises a potential issue under the labeling provisions of the Federal Food, Drug, and Cosmetic Act (FD&C Act). Under section 403(a) of the FD&C Act, a food is misbranded if its labeling is false or misleading in any particular. Section 403(r) of the FD&C Act lays out the statutory framework for the use of labeling claims that characterize the level of a nutrient in a food or that characterize the relationship of a nutrient to a disease or health-related condition. If products that contain B. breve strain M-16V bear any claims on the label or in labeling, such claims are the purview of the Office of Nutrition, Labeling, and Dietary Supplements (ONLDS) in the Center for Food Safety and Applied Nutrition (CFSAN). The Office of Food Additive Safety (OFAS) neither consulted with ONLDS on this labeling issue nor evaluated the information in your notice to determine whether it would support any claims made about B. breve strain M-16V on the label or in labeling.

Allergen Labeling

The Food Allergen Labeling and Consumer Protection Act of 2004 (FALCPA) amends the FD&C Act to require that the label of a food that is or contains an ingredient that bears or contains a “major food allergen” declare the presence of the allergen (section 403(w)). FALCPA defines a “major food allergen” as one of eight foods or food groups (i.e., milk, eggs, fish, Crustacean shellfish, tree nuts, peanuts, wheat, and soybeans) or a food ingredient that contains protein derived from one of those foods. B. breve strain M-16V grown in a fermentation medium that contains milk-based ingredients may require labeling under FALCPA, because it may contain protein derived from milk. Although issues associated with labeling food are the responsibility of ONLDS, issues associated with FALCPA exemptions are the responsibility of the Food Allergen Coordinator in OFAS.

Intended Use in Infant Formula

Under section 412 of the 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. Morinaga should be aware that FDA’s response to Morinaga’s GRAS notice does not alleviate the responsibility of any infant formula manufacturer who intends to market an infant formula that contains B. breve strain M-16V to make the submission required by section 412.

Section 301(ll) of the 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 Morinaga’s notice that B. breve strain M-16V is GRAS for the intended use, FDA did not consider whether section 301(ll) or any of its exemptions apply to foods containing B. breve strain M-16V. Accordingly, this response should not be construed to be a statement that foods that contain B. breve strain M-16V, if introduced or delivered for introduction into interstate commerce, would not violate section 301(ll).


Based on the information provided by Morinaga, as well as other information available to FDA, the agency has no questions at this time regarding Morinaga’s conclusion that B. breve strain M-16V 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 B. breve strain M-16V. As always, it is the continuing responsibility of Morinaga 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 000454, 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)Recommended International Code of Practice, General Principles of Food Hygiene, CAC/RCP 1-1969, Revision 4-2003.

(2)FDA notes that the amount (in grams) of powdered formula per 100 mL reconstituted formula is not a constant value and varies slightly between different infant formulas.

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