[Federal Register Volume 66, Number 95 (Wednesday, May 16, 2001)]
[Rules and Regulations]
[Pages 27020-27022]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 01-12225]


=======================================================================
-----------------------------------------------------------------------

DEPARTMENT OF HEALTH AND HUMAN SERVICES

Food and Drug Administration

21 CFR Part 173

[Docket No. 92F-0396]


Secondary Direct Food Additives Permitted in Food for Human 
Consumption; Alpha-Acetolactate Decarboxylase Enzyme Preparation

AGENCY: Food and Drug Administration, HHS.

ACTION: Final rule.

-----------------------------------------------------------------------

SUMMARY: The Food and Drug Administration (FDA) is amending the food 
additive regulations to provide for the safe use of alpha-acetolactate 
decarboxylase (-ALDC) enzyme preparation derived from Bacillus 
subtilis, modified by recombinant deoxyribonucleic acid (DNA) 
techniques to contain the gene coding for -ALDC from B. 
brevis, for use as a processing aid to produce alcoholic malt beverages 
and distilled liquors. This action is in response to a petition filed 
by Novozymes North America, Inc. (formerly Novo Nordisk Bioindustrials, 
Inc.).

DATES: This rule is effective May 16, 2001. Submit written objections 
and requests for a hearing by June 15, 2001. The Director of the Office 
of the Federal Register approves the incorporation by reference in 
accordance with 5 U.S.C. 552(a) and 1 CFR part 51 of a certain 
publication in Sec. 173.115(b)(3), effective as of May 16, 2001.

ADDRESSES: Submit written objections to the Dockets Management Branch 
(HFA-305), Food and Drug Administration, 5630 Fishers Lane, rm. 1061, 
Rockville, MD 20852.

FOR FURTHER INFORMATION CONTACT: Rudaina H. Alrefai, Center for Food 
Safety and Applied Nutrition (HFS-206), Food and Drug Administration, 
200 C St. SW., Washington, DC 20204, 202-418-3034.

SUPPLEMENTARY INFORMATION: In a notice published in the Federal 
Register of November 30, 1992 (57 FR 56585), FDA announced that a food 
additive petition (FAP 2A4345) had been filed by Novo Nordisk 
Bioindustrials, Inc., later renamed Novozymes North America, Inc., 77 
Perry Chapel Church Rd., P.O. Box 576, Franklinton, NC 27525. The 
petition proposed that the food additive regulations be amended to 
provide for the safe use of -acetolactate decarboxylase (ALDC) 
derived from B. subtilis modified by recombinant DNA techniques to 
contain the gene coding for ALDC from B. brevis for use as a processing 
aid in the brewing and alcohol industries.
    When FDA filed the petition in the Federal Register of November 30, 
1992 (57 FR 56585), it contained an environmental assessment (EA). The 
notice of filing stated `` * * * if the agency finds that an 
environmental impact statement is not required and this petition 
results in a regulation, the notice of availability of the agency's 
finding of no significant impact and the evidence supporting that 
finding will be published with the regulation * * *.'' In the Federal 
Register of July 29, 1997 (62 FR 40570), FDA published a final rule on 
its National Environmental Policy Act policies and procedures, which 
became effective on August 28, 1997. In a letter dated January 4, 2001, 
the petitioner submitted a claim of categorical exclusion under 21 CFR 
25.32(k). The agency has reviewed the claim of categorical exclusion 
and has concluded that it is warranted.

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

A. Introduction

    The use of -ALDC enzyme preparation from B. subtilis is to 
prevent the formation of diacetyl that causes unpleasant taste in beer 
and other alcoholic beverages. The enzyme -ALDC is to be 
distinguished from the -ALDC enzyme preparation, which 
contains -ALDC as the principal active component in addition 
to other components derived from the production organism and 
fermentation media. This document will refer to the former as 
``-ALDC'' and the latter as ``-ALDC enzyme 
preparation.'' Diacetyl is normally formed from -acetolactate 
during fermentation. Alpha-ALDC, which is the active component of the 
petitioned enzyme preparation, catalyzes the conversion of -
acetolactate directly to acetoin, thereby reducing the time needed for 
spontaneous degradation of diacetyl to acetoin.

B. Host Organism

    The host organism, B. subtilis, for production of -ALDC is 
widely distributed in nature and is commonly present in foods eaten by 
both humans and animals. It also has a history of safe use as a source 
of enzymes in food enzyme manufacturing industry prior to 1958. Thus, 
B. subtilis is considered to be a nonpathogenic microorganism.

C. Donor Organism

    B. brevis is the microorganism used as the source of the genetic 
material for the -ALDC enzyme that is the subject of FAP 
2A4345. FDA reviewed the safety of the DNA that encodes the enzyme 
-ALDC from B. brevis and the enzyme it produces (discussed 
below), because only that DNA is transferred to the host strain from 
the donor organism.

D. Production Organism

    The petitioner provided information demonstrating that the plasmid 
carrying the gene for -ALDC is stably integrated into the 
chromosome of B. subtilis production strain. The petitioner conducted a 
study to evaluate the pathogenicity of three B. subtilis strains. In 
this study, mice received an intraperitoneal injection with the B. 
subtilis host strain, B. subtilis production strain, and a B. subtilis 
strain capable of producing -ALDC but not used as a source of 
the petitioned enzyme preparation. FDA reviewed this study as well as 
the scientific literature concerning potential pathogenicity of B. 
subtilis and did not identify any microbiological concern (Refs. 1, 2, 
and 3).

E. Enzyme Preparation

    The -ALDC enzyme preparation is manufactured by a 
submerged pure culture fermentation of a genetically engineered strain 
of B. subtilis carrying the B. brevis gene that encodes -ALDC. 
The enzyme is secreted to the fermentation broth and processed by 
removing the cellular debris, followed by concentration and 
formulation. For certain applications, the -ALDC enzyme 
preparation is stabilized by crosslinking with glutaraldehyde (referred 
to as d-ALDC).
    The petitioner submitted several toxicological studies that address 
the safety of the petitioned -ALDC and d-ALDC enzyme 
preparations. These include: A teratogenicity study in rats and 
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 -ALDC

[[Page 27021]]

enzyme preparation does not raise any toxicity concerns at the expected 
level of consumption or have any mutagenic potential (Refs. 4 and 5).

F. 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 B. subtilis will introduce impurities that raise concerns 
about the safety of the enzyme preparation.
    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., -ALDC), thereby contaminating the 
enzyme preparation. As a matter of current good manufacturing practice, 
manufacturers using recombinant DNA technology must ensure that they 
have not inadvertently cloned extraneous protein-encoding DNA along 
with the -ALDC gene that may lead to contamination of the 
-ALDC 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 -ALDC gene, used in 
the development of the recombinant B. subtilis was accurately 
constructed. The petitioner submitted evidence to demonstrate that it 
cloned a full-length copy of the -ALDC gene from B. brevis 
into B. subtilis. The petitioner also described the multistep process 
for constructing the B. subtilis production strain. These steps involve 
the use of several plasmids (intermediate plasmids) that confer 
resistance to chloramphenicol and kanamycin, both of which are 
clinically useful antibiotics. Through various techniques, these 
plasmids are eliminated during the construction of the gene encoding 
-ALDC. The petitioner tested the final enzyme preparation for 
the presence of the production strain or other microbial activity and 
for antibacterial activity and reported none present. Therefore, the 
agency concludes that the production strain is effectively removed by 
the enzyme purification procedure. Furthermore, the -ALDC 
enzyme preparation conforms to the general and additional requirements 
for enzyme preparations in the Food Chemicals Codex (Ref. 6) and does 
not contain the production organism or antimicrobial activity.
    FDA concludes that, when the -ALDC enzyme preparation is 
manufactured in conformity with Sec. 173.115, there is no basis for 
concern regarding the possibility that the -ALDC enzyme 
preparation will be contaminated by the products of extraneous genetic 
material inserted along with the -ALDC gene in B. subtilis 
(Ref. 1). Furthermore, FDA concludes, having considered the evidence 
concerning the production organism and the processing steps to derive 
the -ALDC enzyme preparation, that: (1) B. subtilis containing 
-ALDC gene from B. brevis is safe for use as a source of food-
grade -ALDC enzyme preparation, (2) impurities resulting from 
the use of B. subtilis containing -ALDC gene from B. brevis in 
the production of -ALDC enzyme preparation will not affect the 
safety of the -ALDC enzyme preparation, and (3) processing 
aids and their impurities that are used to make the commercial 
-ALDC enzyme preparation and that may remain in food processed 
with this enzyme preparation present no safety concerns (Refs. 5 and 
7).

II. Conclusion

    FDA has evaluated the data in the petition and other relevant 
material. Based on this information, the agency concludes that: (1) The 
proposed use of -ALDC enzyme preparation from B. subtilis 
containing the -ALDC gene from B. brevis is safe, (2) the 
additive will achieve its intended technical effect, and (3) the 
regulations in Sec. 173.115 should be amended as set forth below in 
this document.

III. Inspection of Documents

    In accordance with Sec. 171.1 (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 determined under 21 CFR 25.32(k) that this action is 
of a type that does not individually or cumulatively have a significant 
effect on the human environment. Therefore, neither an EA nor an 
environmental impact statement is required.

V. Paperwork Reduction Act of 1995

    This final rule contains no collection of information. Therefore, 
clearance by the Office of Management and Budget under the Paperwork 
Reduction Act of 1995 is not required.

VI. Objections

    Any person who will be adversely affected by this regulation may at 
any time file with the Dockets Management Branch (address above) 
written objections by June 15, 2001. 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 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 are to be submitted and are to 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.

VII. 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. Memorandum from W. Koch, FDA, to L. Kahl, FDA, September 23, 
1993.
    2. Memorandum from J. Madden, FDA, to the Biotechnology Policy 
Branch, FDA, August 29, 1995.

[[Page 27022]]

    3. Memorandum from J. Madden, FDA, to the Biotechnology Policy 
Branch, FDA, October 20, 1995.
    4. Memorandum from R. D. Benz, FDA, to K. C. Raffaele, FDA, July 
20, 1995.
    5. Memorandum from the Division of Health Effects Evaluation, 
FDA, to R. H. Alrefai, FDA, May 4, 1999.
    6. Food Chemicals Codex, 1996, 4th ed., National Academy Press, 
Washington, DC, pp. 133-134.
    7. Memorandum from the Division of Health Effects Evaluation, 
FDA, to R. H. Alrefai, FDA, August 20, 1999.

List of Subjects in 21 CFR Part 173

    Food additives, Incorporation by reference.

    Therefore, under the Federal Food, Drug, and Cosmetic Act and under 
authority delegated to the Commissioner of Food and Drugs, 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 173.115 is added to subpart B to read as follows:


Sec. 173.115  Alpha-acetolactate decarboxylase (]-ALDC) enzyme 
preparation derived from a recombinant Bacillus subtilis.

    The food additive alpha-acetolactate decarboxylase (-ALDC) 
enzyme preparation, may be safely used in accordance with the following 
conditions:
    (a) The food additive is the enzyme preparation derived from a 
modified Bacillus subtilis strain that contains the gene coding for 
-ALDC from Bacillus brevis.
    (b)(1) The manufacturer produces the additive from a pure culture 
fermentation of a strain of Bacillus subtilis that is nonpathogenic and 
nontoxigenic in man or other animals.
    (2) The manufacturer may stabilize the enzyme preparation with 
glutaraldehyde or with other suitable approved food additives or 
generally recognized as safe substances.
    (3) The enzyme preparation must meet the general and additional 
requirements for enzyme preparations in the Food Chemicals Codex, 4th 
ed., 1996, pp. 133-134, which is incorporated by reference. The 
Director of the Office of the Federal Register approves this 
incorporation by reference in accordance with 5 U.S.C. 552(a) and 1 CFR 
part 51. Copies may be obtained from the National Academy Press, 2101 
Constitution Ave. NW., Washington, DC 20055, or may be examined at the 
Center for Food Safety and Applied Nutrition, 200 C St. SW., rm. 3321, 
Washington, DC, or at the Office of the Federal Register, 800 North 
Capitol St. NW., suite 700, Washington, DC.
    (c) The additive is used in an amount not in excess of the minimum 
required to produce its intended effect as a processing aid in the 
production of alcoholic malt beverages and distilled liquors.

    Dated: May 4, 2001.
Margaret M. Dotzel,
Associate Commissioner for Policy.
[FR Doc. 01-12225 Filed 5-15-01; 8:45 am]
BILLING CODE 4160-01-F