Agency Response Letter GRAS Notice No. GRN 000462
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CFSAN/Office of Food Additive Safety
June 6, 2013
3550 John Hopkins Ct.
Re: GRAS Notice No. GRN 000462
Dear Ms. Shanahan:
The Food and Drug Administration (FDA) is responding to the notice, dated February 21, 2013, that you submitted on behalf of Verenium Corporation (Verenium) 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 February 22, 2013, filed it on March 04, 2013, and designated it as GRAS Notice No. GRN 000462.
The subject of the notice is lipase enzyme preparation from modified Pseudomonas fluorescens Biovar I (lipase enzyme preparation). The notice informs FDA of the view of Verenium that lipase enzyme preparation is GRAS, through scientific procedures, as an enzyme in the production of refined triacylglycerol-based oils used in food, at specified levels.
Commercial enzyme preparations that are used in food contain an enzyme component, which catalyzes the chemical reaction 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, Verenium provides information about all the components of lipase enzyme preparation.
According to the classification system of enzymes established by the International Union of Biochemistry and Molecular Biology, lipase is identified by the Enzyme Commission number 22.214.171.124. The accepted name for the enzyme is triacylglycerol lipase and the systematic name is triacylglycerol acylhydrolase. The CAS Registry Number for lipase is 9001-62-1 and the calculated molecular weight of this 227 amino acid protein is 24.45 kiloDaltons. Lipase catalyzes the hydrolysis of triacylglycerol to diacylglycerol and a free fatty acid.
Verenium describes the development of the production strain, P. fluorescens BD29241 (referred to as BD29241) from the host strain, P. fluorescens DC454 (referred to as DC454). P. fluorescens is a common saprophyte, i. e., it grows on dead or decaying organic matter. P. fluorescens is nonpathogenic and nontoxigenic to humans with a history of safe industrial use. DC454 is derived from P. fluorescens Biovar I, MB101 (referred to as MB101). MB101 was identified and verified as P. fluorescens Biotype A, a close relative of Biotype A of P. fluorescens ATCC 17397. Also, a derivative of MB101 was used as a recipient in a previous GRAS Notice (GRN 000126).
Verenium states that BD29241 was constructed by electroporation of DC454 with the expression vector pDOW1169_BD29241 carrying the lipase gene. Verenium used the pyrF gene as a selectable marker in the transformation. Prior to the transformation, DC454 was generated by deleting the pyrF gene, followed by the insertion of the lacIQ1 gene using site-specific mutagenesis. The native lipase gene was obtained from a soil environmental library in E. coli. It was modified to improve palmitic acid selectivity, codon optimized for expression in P. fluorescens, and inserted into DC454. The generation of the expression vector containing the modified lipase gene was also described in GRN000126, when it was used to express alpha-amylase in a P. fluorescens system. Verenium confirmed insertion of pDOW1169 by DNA sequence analysis. Verenium states that the introduced plasmid vector is stable during fermentation, is poorly mobilizable, and is not self-transmissible. Using DNA sequencing, Verenium confirmed that the production strain does not contain antibiotic resistance genes.
Verenium states that the lipase enzyme is produced by a submerged fed-batch fermentation of a pure culture of the P. fluorescens production strain BD29241. During fermentation, the lipase enzyme is expressed intracellularly in the production strain. The cells are lysed using alkaline pH treatment, and the fermentation broth is removed to a dilution tank for enzyme recovery. The broth is then diluted with water and separated from the cell mass with the aid of appropriate food-grade raw materials. The clarified filtrate is then concentrated by ultrafiltration, and the liquid enzyme product is stabilized to desired enzyme activity using appropriate stabilizers and preservatives. The final enzyme preparation is stored in containers held in a temperature controlled facility at 4-8°C. Verenium states that the lipase enzyme preparation is produced in accordance with current good manufacturing practice. Verenium states that components of the fermentation media are not derived from major food allergens; raw materials used in the fermentation, recovery, and formulation meet predefined quality standards and are food grade. Tests confirm the absence of both transformable recombinant DNA and the production microbe in the final lipase enzyme preparation.
Verenium notes that lipase enzyme preparation conforms to the specifications for enzyme preparations described in the Food Chemicals Codex (8th edition, 2012), and to the General Specifications and Considerations for Enzyme Preparations Used in Food Processing established by the FAO/WHO Joint Expert Committee on Food Additives (2006).
Verenium intends to use lipase enzyme preparation containing up to 506 milligrams (mg) per kilogram (kg) of the active enzyme, to reduce palmitic acid levels in the production of refined triacylglycerol-based oils. The refining process of triacylglycerol-based oils includes steps to inactivate and denature the enzyme. Verenium anticipates that the action of lipase enzyme will not result in the formation of any reaction products that are not already part of the human diet. Using a validated ELISA method, Verenium demonstrates levels below the limit of quantitation (< 60 micrograms per kg) of residual enzyme in the final food (degummed and refined bleached and deodorized (RBD) oil). However, to estimate maximum possible dietary intake, Verenium assumes that 100% of the enzyme used in the intended food applications would remain in the finished foods. Based on this assumption, Verenium calculates the maximum daily intake of lipase enzyme preparation from all intended food applications, in terms of Total Organic Solids (TOS) to be 3.54 mg TOS per kg bodyweight per day (mg TOS/kg bw/d) on a per capita basis.
Verenium summarizes published acute, inhalation, 90-day toxicity studies, and unpublished genotoxic studies conducted with the lipase enzyme. The results of the genotoxicity studies including chromosomal aberrations in human lymphocytes, mouse micronucleus assay, and Ames assay showed that the enzyme concentrate is not clastogenic to cultured human lymphocytes, does not increase the induction of mutation frequency, and is not mutagenic, respectively. In the 90-day oral toxicity study in rats, Verenium demonstrates that consumption of the lipase enzyme did not result in treatment-related effects at 1668 mg TOS/kg bw/day, the highest dose tested. Based on the highest dose tested from this study and the maximum daily intake of the notified lipase enzyme preparation, Verenium calculates a margin of safety to be 471, expressed as TOS. Further, Verenium calculates a margin of safety of 4x106 based on the ELISA method developed to detect the enzyme in the final degummed and RBD oil.
Verenium discusses potential food allergenicity of lipase enzyme. Verenium conducted an amino acid sequence homology search using the FASTA software for the notified lipase enzyme against known allergens in the current release of FAARP database, a publicly available database used to predict overall structural similarities with known allergenic proteins. No identity matches of at least 35% over an 80 amino acid segment were found, Therefore, Verenium considers it unlikely that the enzyme will produce an allergenic response via the oral route and concludes that the notified lipase enzyme is not a food allergen.
Based on the data and information summarized above, Verenium concludes that lipase enzyme preparation is GRAS for its intended uses.
Potential Labeling Issues
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. In describing the technical effect of lipase enzyme preparation for its intended use in food, i. e. to reduce palmitic acid levels in the production of physically refined triacylglycerol-based oils, Verenium raises a potential issue under the labeling provisions of the Federal Food, Drug, and Cosmetic Act (FD&C Act). If products that contain lipase enzyme preparation bear any claims on the label or in labeling, such claims are the purview of the Office of Nutrition Labeling and Dietary Supplements (ONLDS). The Office of Food Additive Safety neither consulted with ONLDS nor evaluated the information in your notice to determine whether it would support any claims made about production of physically refined triacylglycerol-based oils using lipase enzyme preparation, on the label or in labeling.
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 Verenium’s notice that lipase 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 lipase enzyme preparation. Accordingly, this response should not be construed to be a statement that foods that contain lipase enzyme preparation, if introduced or delivered for introduction into interstate commerce, would not violate section 301(ll).
Based on the information provided by Verenium, as well as other information available to FDA, the agency has no questions at this time regarding Verenium’s conclusion that lipase 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 lipase enzyme preparation. As always, it is the continuing responsibility of Verenium 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 000462, 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