U. S. Food and Drug Administration
Center for Food Safety and Applied Nutrition
Office of Food Additive Safety
April 17, 2003

Nancy Schnell
Deputy General Counsel - Marketing and Regulatory
Good Humor-Breyers, a Unilever Company
390 Park Avenue
New York, NY 10036

Re: GRAS Notice No. GRN 000117

Dear Ms. Schnell:

The Food and Drug Administration (FDA) is responding to the notice, dated October 18, 2002, 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 October 23, 2002, filed it on October 30, 2002, and designated it as GRAS Notice No. GRN 000117.

The subject of the notice is ice structuring protein (ISP) preparation from Saccharomyces cerevisiae carrying a gene encoding an ISP derived from ocean pout (ISP preparation). The notice informs FDA of the view of Good Humor-Breyers, a Unilever company (Unilever) that ISP preparation is GRAS, through scientific procedures, for use as a texturizer in frozen novelty desserts to provide ISP at a level of 0.01 percent in the finished product.

Unilever describes generally available information about ice-structuring proteins (ISPs). ISPs are naturally occurring proteins found in a wide variety of species that bind to and influence the growth and structure of ice crystals. The ISP considered in this GRAS notice was originally identified in ocean pout, Macrozoarces americanus. There are at least 12 different types of ISPs that have been found in the serum of ocean pout and separated by high performance liquid chromatography (HPLC). Unilever provides detailed information about one of these HPLC-purified ISPs - i.e., ISP type III HPLC 12. This protein is made up of 66 amino acids of known sequence, has a molecular weight of approximately 7 kDa, a pH stability range of 2-12, and is heat tolerant. It is a globular protein with short beta strands and hydrophobic patches, and it is normally not glycosylated. It is identified by the accession number P19614 in the Swiss Prot protein database.

Unilever notes that it would not be practicable to obtain sufficient quantities of ISP type III HPLC 12 for use in frozen novelty desserts directly from ocean pout. In order to obtain large quantities of a protein with the same amino acid sequence as ISP type III HPLC 12, Unilever developed a bioengineered strain of Saccharomyces cerevisiae that contains a synthetic gene encoding ISP type III HPLC 12. Unilever discusses its reasons for concluding that the production strain, S. cerevisiae VWk19 gal1, leu2, pmt1 pUR3993, is genetically stable.

Unilever describes the manufacture of ISP preparation, stating that it is produced using standard industrial-scale biotechnology processes and standard food ingredients. The protein is secreted from the yeast cells into the growth medium. The yeast cells are then separated from the medium by filtration, and the ISP is concentrated and purified by ultrafiltration. The final ISP preparation derived from this process is a mixture of ISP type III HPLC 12, glycosylated ISP, proteins and peptides from the yeast used to produce ISP, and sugars, acids, and salts that are commonly found in food. Unilever provides information about these components as well as specifications for ISP preparation.

Unilever discusses dietary exposure to the ISP type III HPLC 12 protein. Based on publicly available consumption data for frozen novelty desserts, Unilever estimates the highest single-day exposure to be 0.33 milligrams per kilogram body weight per day in males aged 13 to 20 years old. Unilever also provides estimates for average monthly and yearly ISP consumption in frozen desserts of 0.092 milligrams per kilogram body weight per day and 0.061 milligrams per kilogram body weight per day, respectively.

In evaluating the safety of ISP preparation, Unilever considers that the main safety issue to be addressed is potential allergenicity of ISP type III HPLC 12. Unilever addressed this potential from three perspectives. These are (1) the safety of ISP type III HPLC 12 for individuals who are allergic to fish; (2) the safety of ISP preparation (which contains yeast-derived components) for individuals who are sensitive to yeast; and (3) the safety of ISP preparation for the general population. Unilever considers that the approach that it used to address this potential issue is consistent with recent international consensus documents (Refs. 1 and 2).

To assess the potential that ISP type III HPLC 12 is one of the allergenic components of ocean pout, Unilever discusses published and unpublished studies conducted in vitro or in vivo. These studies include assays for binding of immunoglobulin E (IgE) through radioallergosorbent test (RAST), RAST inhibition, immunoblotting, and basophil histamine release, as well as skin prick testing. Unilever conducted these tests using ISP preparation, which contains yeast-derived components. To distinguish between reactions to ISP itself and to the yeast components, these studies included as additional test articles purified ISP type III HPLC 12 protein and a control preparation of yeast extract from the host yeast strain used to develop the production strain. Based on the totality of information from these studies, Unilever concludes that ISP preparation does not present a risk of eliciting allergic reactions in the fish allergic population.

To assess the safety of ISP preparation for individuals who are sensitive to yeast, Unilever discusses the following data and information: (1) Sensitization to yeast appears to be relatively common, particularly in persons who have experienced inhalation exposure, in persons who have experienced a yeast infection, or in persons with atopic dermatitis. This sensitization can be measured by highly sensitive tests such as skin prick tests and RASTs. (2) Allergic responses to yeast are extremely rare, and serious allergic reactions to yeast are almost unknown. This is despite the fact that yeast is ubiquitous in the food supply and has been safely used as a food ingredient throughout recorded history. (3) All of the persons in Unilever's studies who experienced positive RAST and skin prick test responses to yeast also had atopic dermatitis or related conditions. Significantly, all of these individuals are able to eat yeast-containing foods (such as bread, wine, and beer) without adverse reaction. Based on the totality of information, Unilever concludes that the yeast component of the ISP preparation does not pose a clinically significant allergenic risk.

To assess the potential that ISP could induce an allergic response in the general population, Unilever discusses the following information: (1) a published study comparing the amino acid sequence of ISP to that of known allergens; (2) a published study evaluating the ability of ISP to be digested by the gastric enzyme pepsin; and (3) an unpublished human study to look for antibody response to ingestion. Based on the weight of the evidence, Unilever concludes that there is little likelihood that ISP will be allergenic to the general population when ingested.

In addition to these safety studies that were designed to assess the allergenic potential of ISP, Unilever describes unpublished genotoxicity tests and a subchronic rat toxicity study. Unilever concludes that these studies provide no evidence of genotoxicity or other toxicity.

GRN 000117 includes the findings of a panel of individuals (Unilever's GRAS panel) who evaluated the data and information that are the basis for Unilever's GRAS determination. Unilever considers the members of its GRAS panel to be qualified by scientific training and experience to evaluate the safety of substances added to food. Unilever's GRAS panel discusses the identity of ISP preparation; its manufacture, specifications, and exposure; and potential allergenicity, toxicity, and genotoxicity. Unilever's GRAS panel concludes that ISP preparation is safe for individuals who are allergic to fish, for individuals who have been sensitized to yeast, and for the population as a whole. In a meeting on January 23, 2003, between FDA and representatives of Unilever, members of Unilever's GRAS panel with experience in food allergy reiterated these conclusions.

Based on the information provided by Unilever, as well as other information available to FDA, the agency has no questions at this time regarding Unilever's conclusion that ISP 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 ISP preparation. As always, it is the continuing responsibility of Unilever 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, as well as a copy of the information in your notice that conforms to the information in proposed 21 CFR 170.36(c)(1), is available for public review and copying on the homepage of the Office of Food Additive Safety (on the Internet at http://www.cfsan.fda.gov/~lrd/foodadd.html).

References

1 FAO/WHO (2001). Evaluation of allergenicity of genetically modified foods. Report of a Joint FAO/WHO Expert Consultation on Allergenicity of Foods Derived from Biotechnology, Rome, January 22-25, 2001.

2 Codex Alimentarius Commission (2002). Report of the third session of the Codex ad hoc Intergovernmental Task Force on Foods Derived from Biotechnology. (available at http://www.codexalimentarius.net/reports.asp as ALINORM 03/34)