[Federal Register Volume 62, Number 212 (Monday, November 3, 1997)]
[Rules and Regulations]
[Pages 59281-59284]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 97-29048]


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DEPARTMENT OF HEALTH AND HUMAN SERVICES

Food and Drug Administration

21 CFR Part 173

[Docket No. 93F-0461]


Secondary Direct Food Additives Permitted in Food for Human 
Consumption; Milk-Clotting Enzymes

AGENCY: Food and Drug Administration, HHS.

ACTION: Final rule.

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SUMMARY: The Food and Drug Administration (FDA) is amending the food 
additive regulations to provide for the safe use of aspartic proteinase 
enzyme preparation produced by pure culture fermentation of Aspergillus 
oryzae modified by recombinant deoxyribonucleic (DNA) techniques to 
contain the gene for aspartic proteinase enzyme from Rhizomucor miehei 
for use as a milk-clotting enzyme in the production of cheese.

DATES: The regulation is effective November 3, 1997; written objections 
and requests for a hearing by December 3, 1997.

ADDRESSES: Submit written objections to the Dockets Management Branch 
(HFA-305), Food and Drug Administration, 12420 Parklawn Dr., rm. 1-23, 
Rockville, MD 20857.

FOR FURTHER INFORMATION CONTACT: Wendy J. Dixon, Center for Food Safety 
and Applied Nutrition (HFS-206), Food and Drug Administration, 200 C 
St. SW., Washington, DC 20204, 202-418-3090.

SUPPLEMENTARY INFORMATION:
    In a notice published in the Federal Register of January 21, 1994 
(59 FR 3365), FDA announced that a food additive petition (FAP 4A4406) 
had been filed by Novo Nordisk Bioindustrials, Inc., proposing that the 
food additive regulations be amended to provide for the safe use of 
aspartic proteinase enzyme preparation produced by pure culture 
fermentation of A. oryzae modified by recombinant DNA techniques to 
contain the gene for aspartic proteinase enzyme from R. miehei for use 
in the production of cheese. Although Novo Nordisk Bioindustrials, 
Inc., submitted FAP 4A4406, while the petition was under review, Gist-
Brocades International B. V. purchased the dairy enzyme business from 
Novo Nordisk, at which time, the responsibility for the petition 
transferred to Gist-Brocades International B. V.

[[Page 59282]]

I. Evaluation of Safety of the Petitioned Use of the Additive

A. Aspergillus Oryzae

    The host organism for production of aspartic proteinase is the 
fungus A. oryzae. A. oryzae has had a long history of use, greater than 
2,000 years, in the production of enzymes, e.g., koji and <greek-a>-
amylase, used in the fermentation and processing of food products, such 
as soy-sauce, miso, sake, baked goods, and brewery products (Refs. 1 
and 2). The nonpathogenicity and nontoxigenicity of this microbe to 
humans and its inability to produce antibiotics is well-documented in 
the literature (Refs. 1, 3, and 4). This conclusion regarding the 
nonpathogenicity and nontoxigenicity of this microbe is consistent with 
a recent evaluation of the Joint Expert Committee on Food Additives 
(JECFA) of the Food and Agriculture Organization (FAO) and the World 
Health Organization (WHO) (Ref. 5). JECFA evaluated the current uses of 
A. oryzae and enzyme preparations therefrom and concluded that the 
amylases and proteases from A. oryzae that were included in JECFA's 
review should be regarded as foods and thus, are safe for use in food 
processing.
    The petitioner submitted a study to investigate the pathogenic 
potential of five strains of A. oryzae, including the parental strain 
and four recombinant strains; one of the strains tested is the subject 
of this petition. FDA evaluated this study and concluded that the 
recombinant strains of A. oryzae, as well as the unmodified parental 
strain, demonstrated no pathogenicity for mice when spores were 
inoculated in large numbers. Previously, A. oryzae has been the subject 
of evaluations performed by FDA, and based on those evaluations FDA 
concluded that the spores of two strains of A. oryzae are nonpathogenic 
to mice (Ref. 6). Therefore, FDA concludes that the recombinant strain 
of A. oryzae that is the subject of this petition is nonpathogenic and 
nontoxigenic (Ref. 3).

B. Rhizomucor Miehei

     R. miehei, originally named Mucor miehei (Ref. 7), is the 
microorganism used as the source of the genetic material for the 
aspartic proteinase enzyme that is the subject of FAP 4A4406. Enzyme 
preparations derived from R. miehei (aspartic proteinase, or esterase-
lipase activity produced by pure culture fermentation of R. miehei (as 
M. miehei)) are food additives that are approved for use in cheese 
production under Secs. 173.150(a)(4) and 173.140 (21 CFR 173.150(a)(4) 
and 173.140), respectively.

C. Aspartic Proteinase Preparation

    As discussed above, aspartic proteinase preparation produced by 
pure culture fermentation of R. miehei for use as a milk-clotting agent 
in the production of cheese is an approved food additive under 
Sec. 173.150(a)(4). The petitioner has submitted the following evidence 
to demonstrate that it has cloned full length copies of the aspartic 
proteinase gene from R. miehei into A. oryzae: (1) DNA sequencing 
information, whereby the cloned putative aspartic proteinase gene was 
shown to have the same nucleotide sequence that encodes the amino acid 
sequence of the R. miehei aspartic proteinase; and (2) nucleic acid 
hybridization studies whereby the cloned DNA fragments were shown to 
hybridize (i.e., specifically bind) with complementary DNA from the 
aspartic proteinase gene.
    To further confirm the identity of the aspartic proteinase cloned 
into A. oryzae, the petitioner provided information on the sodium 
dodecyl polyacrylamide gel electrophoresis (SDS-PAGE)\1\ relative 
mobility of recombinant aspartic proteinase and aspartic proteinase 
from R. miehei, with and without treatment by endo-glucosidase H (an 
enzyme that removes most glycosyl moieties from proteins). The results 
from this study establish that untreated aspartic proteinase from 
recombinant A. oryzae has a lower relative mobility than untreated 
aspartic proteinase from R. miehei. However, after pretreatment with 
endo-glucosidase H, the aspartic proteinase preparations from both 
recombinant A. oryzae and R. miehei have an identical SDS-PAGE relative 
mobility. This is higher than the mobilities of either of the untreated 
forms of aspartic proteinase. These results show that aspartic 
proteinase from A. oryzae or R. miehei is glycosylated but when the R. 
miehei gene for aspartic proteinase is expressed in A. oryzae, the 
aspartic proteinase enzyme is more extensively glycosylated (Ref. 8).
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    \1\ SDS-PAGE is a technique that enables one to compare the 
relative molecular weight of proteins based on their rate of 
migration through the gel. The SDS-PAGE relative mobility of a 
protein is directly related to its molecular weight.
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    FDA finds that glycosylation of the aspartic proteinase enzyme does 
not raise any safety concerns. Glycosylation is characteristic of many 
proteins produced in the cells of eukaryotic organisms, which include 
higher plants and animals, and fungi, such as A. oryzae and R. miehei 
(Ref. 9). However, the type and amount of glycosyl moieties attached to 
glycoproteins varies, even among closely related organisms (Ref. 10). 
Therefore, proteins with identical amino acid sequences may have 
different amounts and types of glycosylation when produced in different 
eukaryotic organisms, such as A. oryzae and R. miehei. Because A. 
oryzae is a common, nonpathogenic, nontoxigenic organism that has a 
safe history of use in the production of food processing enzymes (Refs. 
1 and 3), the agency finds that the more extensively glycosylated 
aspartic proteinase enzyme from recombinant A. oryzae is as safe as the 
less extensively glycosylated aspartic proteinase enzyme from R. 
miehei.
    The petitioner submitted several toxicological studies that address 
the safety of the petitioned aspartic proteinase preparation. These 
include: (1) Short term and subchronic toxicity studies in both rats 
and dogs; (2) a teratogenicity study in rats; and (3) genotoxicity 
studies, including tests for mutagenic activity in Salmonella 
typhimurium and mammalian cells, as well as tests for chromosome-
damaging activity in human lymphocytes. FDA has reviewed these studies 
and concludes that the petitioned aspartic proteinase preparation does 
not raise any toxicity concerns at the expected level of consumption 
nor does it have any mutagenic potential (Refs. 6, 11, and 12).

D. Source of Impurities

    Enzyme preparations used in food are usually not chemically pure, 
but contain cellular and processing material. The nature and amounts of 
these impurities in the finished enzyme preparation depend on the 
organism from which the enzyme preparation is produced (the production 
organism), the fermentation materials and methods used to grow the 
production organism, and the materials and methods used to generate the 
finished enzyme preparation. Thus, the question is whether the 
production organism or the manufacturing methods used to grow the 
production organism or to generate the finished enzyme preparation from 
recombinant A. oryzae, will introduce impurities that raise concerns 
about the safety of the enzyme preparation. In addition, 
Sec. 173.150(c) states that the milk-clotting enzyme preparation shall 
be produced by a process that completely removes the generating 
organism from the milk-clotting enzyme product. The agency concludes 
that the petition contains information demonstrating that the 
manufacturing process includes procedures to ensure that the production 
organism is completely removed from the enzyme preparation

[[Page 59283]]

during the manufacturing (Refs. 3 and 13).
    One issue raised by the use of recombinant DNA techniques is the 
potential transfer of DNA encoding for extraneous proteins along with 
the gene of interest (i.e., aspartic proteinase), thereby contaminating 
the enzyme preparation. As a matter of current good manufacturing 
practice, manufacturers using recombinant DNA technology should ensure 
that they have not inadvertently cloned extraneous protein-encoding DNA 
along with the aspartic proteinase gene that may lead to contamination 
of the aspartic proteinase enzyme preparation. Such assurance can come 
from reviewing the details of the cloning steps, which include the 
origin and sequence of the DNA fragments used in the cloning, and full 
characterization of the final genetic constructs via techniques such as 
DNA sequencing.
    The petition contains information demonstrating that the petitioner 
evaluated the cloning process to ensure that the final cloning product, 
i. e., the DNA with the aspartic proteinase gene and other components 
to ensure accurate expression of the gene, used in the development of 
the recombinant A. oryzae was accurately constructed. As mentioned 
above, the petitioner submitted evidence to demonstrate that it cloned 
full length copies of the aspartic proteinase gene from R. miehei into 
A. oryzae. In addition to the aspartic proteinase gene, the recombinant 
A. oryzae strain contains a marker gene conferring resistance to 
ampicillin (amp<SUP></SUP>r), a clinically useful antibiotic, as well 
as a marker gene encoding the enzyme acetamidase (amdS), which permits 
the transformed strain to utilize acetamide as a nitrogen or carbon 
source. The petitioner states that the only transgenes expressed in the 
production organism, A. oryzae, are the aspartic proteinase transgene 
and the amdS transgene. Aspartic proteinase is secreted into the 
culture medium from which the enzyme preparation is produced while the 
enzyme acetamidase is not. Therefore, the agency concludes that the 
acetamidase is effectively removed when the production cells are 
discarded during processing (Ref. 13).
    The expression of the amp<SUP></SUP>r gene is controlled by a 
promoter, a region of DNA that is a major component in the regulation 
of a gene. In general, bacterial promoters do not function in higher 
organisms, including the fungus A. oryzae. Because expression of the 
amp<SUP></SUP>r gene is controlled by a bacterial promoter, this gene 
is not expected to be expressed in the production organism, A. oryzae. 
The agency has considered the potential consequences if expression of 
the amp<SUP></SUP>r transgene were to occur in the production organism. 
The petitioner noted that the enzyme preparation is produced from the 
fermentation supernatant and that in the process, intact cells are 
removed. Therefore, even if expression of the amp<SUP></SUP>r gene 
takes place, the gene product would be sequestered within the intact 
cells and therefore, would not be present in the fermentation 
supernatant, which is the source of the aspartic proteinase enzyme 
preparation. Accordingly, the agency concludes that any amp<SUP></SUP>r 
gene product would effectively be removed from the enzyme preparation 
(Ref. 13).
    Finally, FDA notes that Sec. 173.150(b) stipulates that the 
microbial milk-clotting enzyme listed in the food additive regulations 
should be produced using a production strain that is nonpathogenic and 
nontoxic in man or other animals. For example, if the DNA inserted by 
recombinant methodology were to encode a toxic substance that would 
render the enzyme preparation unsafe, the resulting aspartic proteinase 
preparation would not conform with the prescribed conditions under 
Sec. 173.150, and therefore, food processed with the improperly 
manufactured enzyme preparation would be deemed adulterated.
    FDA concludes that, when the aspartic proteinase preparation is 
manufactured in conformity with Sec. 173.150, there is no basis for 
concern regarding the possibility that the aspartic proteinase 
preparation will be contaminated by the products of extraneous protein-
encoding DNA (e.g., products of amdS and amp<SUP></SUP>r genes) 
inserted along with the aspartic proteinase gene in A. oryzae (Ref. 
13).
    Furthermore, FDA concludes, having considered the evidence 
concerning the production organism and the processing steps to derive 
the aspartic proteinase preparation, that A. oryzae containing aspartic 
proteinase gene from R. miehei is safe for use as a source of food-
grade aspartic proteinase preparations, and that impurities resulting 
from the use of A. oryzae containing aspartic proteinase gene from R. 
miehei in the production of aspartic proteinase preparation will not 
affect the safety of the aspartic proteinase preparation.

II. Conclusion

    The agency finds that the principal active ingredient, i.e., 
aspartic proteinase, in the aspartic proteinase enzyme preparation, is 
the same as that in the milk-clotting enzyme preparation from R. 
miehei, and that when the preparation is manufactured in accordance 
with the conditions of use listed in Sec. 173.150, the source organism 
and manufacturing process will not introduce impurities that may render 
the use of the enzyme preparation unsafe.
    The agency has evaluated the data in the petition and other 
relevant material. Based on this information, the agency concludes that 
the proposed use of aspartic proteinase enzyme preparation from A. 
oryzae containing the aspartic proteinase gene from R. miehei is safe 
and that the additive will achieve its intended technical effect. 
Therefore, the regulation in Sec. 173.150 should be amended.

III. Inspection of Documents

    In accordance with Sec. 171.1(h) (21 CFR 171.1(h)), the petition 
and the documents that FDA considered and relied upon in reaching its 
decision to approve the petition are available for inspection at the 
Center for Food Safety and Applied Nutrition by appointment with the 
information contact person listed above. As provided in Sec. 171.1(h), 
the agency will delete from the documents any materials that are not 
available for public disclosure before making the documents available 
for inspection.

IV. Environmental Impact

    The agency has carefully considered the potential environmental 
effects of this action. FDA has concluded that the action will not have 
a significant impact on the human environment, and that an 
environmental impact statement is not required. The agency's finding of 
no significant impact and the evidence supporting that finding, 
contained in an environmental assessment, may be seen in the Dockets 
Management Branch (address above) between 9 a.m. and 4 p.m., Monday 
through Friday.

V. Objections

    Any person who will be adversely affected by this regulation may at 
any time on or before December 3, 1997, file with the Dockets 
Management Branch (address above) written objections thereto. Each 
objection shall be separately numbered, and each numbered objection 
shall specify with particularity the provisions of the regulation to 
which objection is made and the grounds for the objection. Each 
numbered objection on which a hearing is requested shall specifically 
so state. Failure to request a hearing for any particular objection 
shall constitute a waiver of the right to a hearing on that objection. 
Each numbered objection for which a hearing is requested shall include 
a detailed description and

[[Page 59284]]

analysis of the specific factual information intended to be presented 
in support of the objection in the event that a hearing is held. 
Failure to include such a description and analysis for any particular 
objection shall constitute a waiver of the right to a hearing on the 
objection. Three copies of all documents shall be submitted and shall 
be identified with the docket number found in brackets in the heading 
of this document. Any objections received in response to the regulation 
may be seen in the Dockets Management Branch between 9 a.m. and 4 p.m., 
Monday through Friday.

VI. References

    The following references have been placed on display in the Dockets 
Management Branch (address above) and may be seen by interested persons 
between 9 a.m. and 4 p.m., Monday through Friday.
    1. Barbesgaard, P., H. P. Heldt-Hansen, and B. Diderichsen, ``On 
the Safety of Aspergillus oryzae: A Review,'' Applied Microbiology 
and Biotechnology, 36:569-572, 1992.
    2. ``Biotechnologies and Food: Assuring the Safety of Foods 
Produced by Genetic Modification,'' Regulatory Toxicology and 
Pharmacology, 12 (3):S114-S128, 1990.
    3. Memorandum from J. Madden, FDA, to D. Keefe, FDA, April 11, 
1994.
    4. Gray, W. D., The Use of Fungi as Food and in Food Processing, 
pp. 42-100, CRC Press, Cleveland, OH, 1970.
    5. Joint FAO/WHO Expert Committee on Food Additives. 
``Toxicological Evaluation of Certain Food Additives,'' 31st 
Meeting, Geneva, February 16-25, 1987.
    6. Memorandum from H. C. A. Chang, FDA, to D. Keefe, FDA, March 
14, 1994.
    7. Shipper, M. A. A., ``On the Genera Rhizomucor and 
Parasitella,'' Studies in Mycology, 17:53-65, 1978.
    8. Cristensen, T. et al., ``High Level Expression of Recombinant 
Genes in Aspergillus oryzae,'' Bio/Technology, 6:1419-1422, 1988.
    9. Herrman, J. L. et al., ``Bacterial Glycoproteins: A Link 
Between Glycosylation and Proteolytic Cleavage of a 19 kDa Antigen 
from Mycobacterium tuberculosis,'' EMBO Journal, 15:3547-3554, 1996.
    10. Grinna, L. S., and J. F. Tschopp, ``Size Distribution and 
General Structural Features of N-linked Oligosaccharides from the 
Methylotrophic Yeast, Pichia pastoris,'' Yeast, 5 (2):107-115, 1989.
    11. Memorandum from S. E. Carberry, FDA, to D. Keefe, FDA, 
January 5, 1995.
    12. Memorandum from H. C. A. Chang, FDA, to D. Keefe, FDA, 
February 6, 1995.
    13. Memorandum from T. A. Cebula, FDA, to D. Keefe, FDA, April 
4, 1995.

List of Subjects in 21 CFR Part 173

    Food additives.
    Therefore, under the Federal Food, Drug, and Cosmetic Act and under 
authority delegated to the Commissioner of Food and Drugs and 
redelegated to the Director, Center for Food Safety and Applied 
Nutrition, 21 CFR part 173 is amended as follows:

PART 173--SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR 
HUMAN CONSUMPTION

    1. The authority citation for 21 CFR part 173 continues to read as 
follows:

    Authority: 21 U.S.C. 321, 342, 348.

    2. Section 173.150 is amended by adding paragraph (a)(5) to read as 
follows:


Sec. 173.150  Milk-clotting enzymes, microbial.

* * * * *
    (a) * * *
    (5) Aspergillus oryzae modified by recombinant deoxyribonucleic 
(DNA) techniques to contain the gene coding for aspartic proteinase 
from Rhizomucor miehei var. Cooney et Emerson as defined in paragraph 
(a)(4) of this section, and classified as follows: Class, 
Blastodeuteromycetes (Hyphomycetes); order, Phialidales (Moniliales); 
genus, Aspergillus; species oryzae.

    Dated: October 20, 1997.
L. Robert Lake,
Director, Office of Policy, Planning and Strategic Initiatives, Center 
for Food Safety and Applied Nutrition.
[FR Doc. 97-29048 Filed 10-31-97; 8:45 am]
BILLING CODE 4160-01-F