[Federal Register: April 23, 1999 (Volume 64, Number 78)]
[Rules and Regulations]
[Page 19887-19895]
From the Federal Register Online via GPO Access [wais.access.gpo.gov]
[DOCID:fr23ap99-13]

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DEPARTMENT OF HEALTH AND HUMAN SERVICES

Food and Drug Administration

21 CFR Part 184

[Docket No. 84G-0257]


Carbohydrase and Protease Enzyme Preparations Derived From
Bacillus Subtilis or Bacillus Amyloliquefaciens; Affirmation of GRAS
Status as Direct Food Ingredients

AGENCY: Food and Drug Administration, HHS.

ACTION: Final rule.

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SUMMARY: The Food and Drug Administration (FDA) is affirming that
carbohydrase enzyme preparations derived from either Bacillus subtilis
or B. amyloliquefaciens and protease enzyme preparations derived from
either B. subtilis or B. amyloliquefaciens are generally recognized as
safe (GRAS) for use as direct food ingredients. This action is a
partial response to a petition filed by the Ad Hoc Enzyme Technical
Committee (now the Enzyme Technical Association).

 DATES: The regulation is effective April 23, 1999. 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 certain
publications listed in 21 CFR 184.1148 and 184.1150, effective April
23, 1999.
FOR FURTHER INFORMATION CONTACT: Linda S. Kahl, Center for Food Safety
and Applied Nutrition (HFS-206), Food and Drug Administration, 200 C
St. SW., Washington, DC 20204, 202-418-3101.

SUPPLEMENTARY INFORMATION:

Table of Contents

I. Introduction
II. Standards for GRAS Affirmation
III. Background
    A.   Identity and Technical Effect
    B.   Methods of Manufacture
IV. Safety Evaluation
    A.   Pre-1958 History of Use in Food
    1.   Bacillus Subtilis
    2.   Bacillus Amyloliquefaciens
    B. Corroborating Evidence of Safety
    1.   The Enzyme Components
    2.   Enzyme Sources, Manufacturing Methods, and Processing Aids
V. Comments
VI. Conclusions
VII. Environmental Considerations
VIII. Analysis for Executive Order 12866
IX. Regulatory Flexibility Analysis
X. Paperwork Reduction Act of 1995
XI. Effective Date
XII. References

I. Introduction

    In accordance with the procedures described in Sec. 170.35 (21 CFR
170.35), the Ad Hoc Enzyme Technical Committee (now the Enzyme
Technical Association), c/o Miles Laboratories, Inc., 1127 Myrtle St.,
Elkhart, IN 46514, submitted a petition (GRASP 3G0016) requesting that
the following enzyme preparations be affirmed as GRAS for use in food:
(1) Animal-derived enzyme preparations: Catalase (bovine liver);
lipase, animal; pepsin; rennet; rennet, bovine; and trypsin; (2) plant-
derived enzyme preparations: Bromelain; malt; and papain; (3)
microbially-derived enzyme preparations: Lipase, catalase, glucose
oxidase, and carbohydrase from Aspergillus niger, var.; mixed
carbohydrase and protease from Bacillus subtilis, var.; carbohydrase
from Rhizopus oryzae; and carbohydrase from Saccharomyces species.
    FDA published a notice of filing of this petition in the Federal
Register of April 12, 1973 (38 FR 9256), and gave interested persons an
opportunity to submit comments to the Dockets Management Branch (HFA-
305), Food and Drug Administration, 5630 Fishers Lane, rm. 1061,
Rockville, MD 20852. The petition was amended by notices published in
the Federal Register of June 12, 1973 (38 FR 15471), proposing
affirmation that microbially-derived enzyme preparations (carbohydrase,
lipase, and protease) from A. oryzae are GRAS for use in food; in the
Federal Register of August 29, 1984 (49 FR 34305), proposing
affirmation that the enzyme preparations ficin, obtained from species
of the genus Ficus (fig tree), and pancreatin, obtained from bovine and
porcine pancreas, are GRAS for use in food; in the Federal Register of
June 23, 1987 (52 FR 23607), proposing affirmation that the protease
enzyme preparation from A. niger is GRAS for use in food; and in the
Federal Register of August 5, 1996 (61 FR 40648), proposing affirmation
that carbohydrase and protease enzyme preparations from B.
amyloliquefaciens are GRAS for use in food. In the June 23, 1987,
notice, FDA also noted the petitioner's assertion that pectinase enzyme
preparation from A. niger and lactase enzyme preparation from A. niger
are included under carbohydrase enzyme preparation from A. niger, and
that invertase enzyme preparation from Saccharomyces cerevisiae and
lactase enzyme preparation from Kluyveromyces marxianus are both
included under carbohydrase enzyme preparation from species of the
genus Saccharomyces. The agency further noted that, therefore,
pectinase enzyme preparation from A. niger, lactase enzyme preparation
from A. niger, invertase enzyme preparation from S. cerevisiae, and
lactase enzyme preparation from K. marxianus were to be considered part
of the petition. Interested persons were given an opportunity to submit
comments to the Dockets Management Branch (address above) on each
amendment.
    After the petition was filed, the agency published, as part of its
comprehensive safety review of GRAS substances, two GRAS affirmation
regulations that covered three of the enzyme preparations from animal
and plant sources included in the petition. These two regulations are:
(1) Sec. 184.1685 Rennet (animal derived) (21 CFR 184.1685), which was
published in the Federal Register of November 7, 1983 (48 FR 51151) and
includes the petitioned enzyme preparations rennet and bovine rennet;
and (2) Sec. 184.1585 Papain (21 CFR 184.1585), which was published in
the Federal Register of October 21, 1983 (48 FR 48805). Thus,

[[Page 19888]]

rennet, bovine rennet, and papain are already affirmed as GRAS and need
not be addressed further.
    In a notice published in the Federal Register of September 20, 1993
(58 FR 48889), the agency announced that the petitioner had requested
that the following enzyme preparations be withdrawn from the petition
without prejudice to the filing of a future petition: (1) Pancreatin
used for its lipase activity, (2) pancreatin used for its amylase
activity, and (3) amylase derived from unmalted barley extract. In that
notice, the agency stated that, in light of the petitioner's request,
any future action by FDA on the petition would not include a
determination of the GRAS status of these three enzyme preparations.
    In a final rule published in the Federal Register of June 26, 1995
(60 FR 32904), the agency affirmed as GRAS the following enzyme
preparations derived from animal sources: Catalase (bovine liver),
animal lipase, pepsin, trypsin, and pancreatin (as a source of protease
activity). In that same final rule, the agency also affirmed as GRAS
the following enzyme preparations derived from plant sources:
Bromelain, ficin, and malt.
    This final rule addresses the following bacterially-derived enzyme
preparations: (1) carbohydrase enzyme preparation from B. subtilis; (2)
protease enzyme preparation from B. subtilis; (3) carbohydrase enzyme
preparation from B. amyloliquefaciens; and (4) protease enzyme
preparation from B. amyloliquefaciens. \1\ The other microbial enzyme
preparations in the petition will be dealt with separately in a future
issue of the Federal Register.
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    \1\ Although the petition requested GRAS affirmation for mixed
carbohydrase and protease enzyme preparation from B. subtilis, the
petitioner subsequently agreed that this enzyme preparation be
evaluated as two separate enzyme preparations, carbohydrase enzyme
preparation from B. subtilis and protease enzyme preparation from B.
subtilis. Enzyme preparations that contain mixtures of carbohydrases
and proteases can be used either for their carbohydrase activity or
for their protease activity, and they are usually sold according to
their intended use. FDA requested the petitioner's agreement to this
change to reflect the distinct uses of mixed carbohydrase and
protease enzyme preparations in food depending on whether a
particular preparation is being used for its carbohydrase activity
or for its protease activity.
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II. Standards for GRAS Affirmation

    Under Sec. 170.30 (21 CFR 170.30) and 21 U.S.C. 321(s), general
recognition of safety may be based only on the views of experts
qualified by scientific training and experience to evaluate the safety
of substances directly or indirectly added to food. The basis of such
views may be either scientific procedures or, in the case of a
substance used in food prior to January 1, 1958, experience based on
common use in food. General recognition of safety based upon scientific
procedures requires the same quantity and quality of scientific
evidence as is required to obtain approval of a food additive and
ordinarily is based upon published studies, which may be corroborated
by unpublished studies and other data and information (Sec. 170.30(b)).
General recognition of safety through experience based on common use in
food prior to January 1, 1958, may be determined without the quantity
or quality of scientific procedures required for approval of a food
additive, and ordinarily is based upon generally available data and
information concerning the pre-1958 use of the substance
(Sec. 170.30(c)).
    For the enzyme preparations from B. subtilis and B.
amyloliquefaciens that are the subject of this document, the Enzyme
Technical Association bases its request for affirmation of GRAS status
on a history of safe food use prior to 1958. As discussed in the
preamble to the proposed rule for the most recent amendment to
Sec. 170.30, general recognition of safety through experience based on
common use in food requires a consensus on the safety of the substance
among the community of experts who are qualified to evaluate the safety
of food ingredients (50 FR 27294 at 27295, July 2, 1985).

III. Background

A. Identity and Technical Effect

    Enzymes are proteins that originate from living cells and produce
chemical change by catalytic action (Random House Dictionary of the
English Language, 1987). Most enzymes are very specific in their
ability to catalyze only certain chemical reactions; this high degree
of specificity and strong catalytic activity are the most important
functional properties of enzymes (Ref. 1).
    Commercial enzyme preparations such as those that are the subject
of this document usually contain several enzymes that have catalytic
activities other than those for which they are sold--i.e., other than
their characterizing enzyme activities. As discussed in more detail in
section III.B of this document, the methods of manufacture for a
specific commercial enzyme preparation are tailored to maximize the
characterizing enzyme activity. The other enzymes that are present in
the preparation generally are present at low levels.
    Carbohydrases, which are also known as glycosidases, are enzymes
whose catalytic activity is the hydrolysis (i.e., splitting) of O-
glycosyl bonds in carbohydrates. The carbohydrase enzyme preparations
that are the subject of this document each contain two or more
carbohydrases, including: (1) <greek-a>-amylase, which hydrolyzes
<greek-a>-1,4-glucan bonds in polysaccharides (e.g., starch) yielding
monosaccharides, linear oligosaccharides and branched oligosaccharides
(dextrins), and (2) <greek-b>-glucanase, which hydrolyzes 1,3 and some
1,4 linkages in <greek-b>-D-glucans (polysaccharides that are common in
cereals such as oats, barley, and rye), yielding oligosaccharides and
glucose (Refs. 2 and 3). Because the major carbohydrase in the
carbohydrase enzyme preparations derived from B. subtilis or B.
amyloliquefaciens is <greek-a>-amylase, the primary use of these enzyme
preparations is the hydrolysis of starch in processes such as the
preparation of starch syrups and the fermentation of beer (Refs. 3
through 5).
    Proteases are enzymes whose catalytic activity is the hydrolysis of
peptide bonds in proteins, yielding peptides and amino acids. The
protease enzyme preparations that are the subject of this document each
contain two or more proteases, including subtilisin and neutral
proteinase (Refs. 2 and 3). The primary use of the protease enzyme
preparations derived from B. subtilis or B. amyloliquefaciens is in the
preparation of protein hydrolysates and the tenderizing of meat (Refs.
3 through 5).
    Table 1 lists the characterizing enzyme activities and associated
International Union of Biochemistry Enzyme Commission (EC) numbers of
the carbohydrase and protease enzyme preparations derived from B.
subtilis or B. amyloliquefaciens.

   #Table 1.--Enzyme Activities and EC Numbers Associated With Enzyme
      Preparations Derived From B. Subtilis or B. Amyloliquefaciens
------------------------------------------------------------------------
                          Characterizing Enzyme
   Enzyme Preparation            Activity                EC Number
------------------------------------------------------------------------
Carbohydrase             <greek-a>-Amylase        3.2.1.1
                         <greek-b>-Glucanase      3.2.1.6
Protease                 Subtilisin               3.4.21.62
                         Neutral Proteinase       3.4.24.28
------------------------------------------------------------------------

B. Methods of Manufacture

    All microbial strains, including bacterial strains, used to
manufacture enzyme preparations are started from a

[[Page 19889]]

pure laboratory culture and grown, or ``fermented,'' in a sterile
liquid nutrient medium or sterile moistened semisolid medium. Accepted
microbiological techniques are used to exclude contaminating organisms
and to avoid development of substrains from within the culture itself
(Ref. 6). Although specific conditions of fermentation vary from
manufacturer to manufacturer, common fermentation procedures are: (1)
The submerged culture method, which uses closed fermenters equipped
with agitators, aeration devices, and jackets or coils for temperature
control; and (2) the semisolid culture method, which uses horizontal
rotating drums or large chambers fitted with trays (Refs. 5 and 6).
During fermentation by either method, the pH, temperature, appearance
or disappearance of certain ingredients, purity of culture, and level
of enzyme activity must be carefully controlled. The fermentation is
harvested at the point where laboratory tests indicate that maximum
production of enzyme activity has been attained.
    In practice, the processes by which microbial-derived enzyme
preparations are produced vary widely. Each single strain of
microorganism produces a large number of enzymes (Ref. 5). The absolute
and relative amounts of various individual enzymes produced vary
markedly among species and even among strains of the same species. They
also vary depending upon the composition of medium on which the
microorganism grows, and upon the fermentation conditions. The
petitioner states that for a specific enzyme preparation the production
strain, medium composition, and fermentation conditions are optimized
to maximize the desired enzyme activity (Refs. 7 and 8).
    The carbohydrase and protease enzymes from B. subtilis and B.
amyloliquefaciens are excreted into the fermentation medium (Refs. 9
through 11). In the semisolid culture method, an enzyme that is present
in the fermentation medium is extracted either directly from the moist
material, or later after the culture mass has been dried. In the
submerged culture method, the microorganisms and other insolubles are
removed from the fermentation medium by decanting, filtering, or
centrifuging, and therefore an extraction step is not required. In
either method, further processing steps may involve clarification,
evaporation, precipitation, drying, and grinding (Refs. 6 and 9 through
12).

IV. Safety Evaluation

A. Pre-1958 History of Use in Food

    Enzyme preparations have been safely used for many years in the
production and processing of food, for example, in the baking, dairy,
and brewing industries (e.g., see Refs. 1, 4, and 13).
1. Bacillus Subtilis
    The petitioner has provided generally available information,
including published reviews, showing that carbohydrase and protease
enzyme preparations derived from B. subtilis were commonly used in food
prior to 1958 (Refs. 4 and 5). This information is summarized in Table
2.

  Table 2.--Applications of Bacterial Carbohydrase and Protease Enzyme
                   Preparations in Food Prior to 1958
------------------------------------------------------------------------
                                          Technical
      Enzyme                              effect or
   preparation       Food categories      industry          References
                                         application
------------------------------------------------------------------------
 Carbohydrase       Beer               Mashing<SUP>1</SUP>          4 and 5
                    Syrup for cocoa    Reduction of      4 and 5
                    and chocolate      viscosity         4 and 5
                    Sugar              Recovery from     4 and 5
                    Distilled          scrap candy       4
                    beverages          Mashing
                    Precooked          Modification of
                    cereals            cereal starches
                                       to improve
                                       characteristics
 Protease           Beer               Chillproofing     4
                    Condiments         Not reported      5
                    Milk               Protein           5
                                       hydrolysis
------------------------------------------------------------------------
\1\ Mashing is the conversion of starch to sugars.

    In the published article by Underkofler et al. (Ref. 5), the
authors use the general terms ``bacterial amylase'' and ``bacterial
protease'' to refer to bacterially-derived carbohydrase and protease
enzyme preparations used in food at the time of the article. However,
the article also includes a table in which the source bacterium for
bacterially-derived enzyme preparations is identified as B. subtilis.
    In the published article by Underkofler and Ferracone (Ref. 4), the
authors use the general terms ``bacterial carbohydrase'' and
``bacterial protease'' to refer to bacterially-derived carbohydrase and
protease enzyme preparations used in food at the time of the article.
Unlike the Underkofler et al. article, however, the Underkofler and
Ferracone article does not identify the source bacterium for these
enzyme preparations. Although it is not possible to determine
conclusively whether the descriptor ``bacterial'' in the Underkofler
and Ferracone article refers to B. subtilis, the use of this term by
the same principal author in two scientific articles published in
consecutive years to describe the source of protease and carbohydrase
enzyme preparations used in the food industry, coupled with the
identification of the source bacterium for these enzyme preparations as
B. subtilis in the Underkofler et al. article, makes it likely that the
source bacterium referred to by Underkofler and Ferracone was in fact
B. subtilis.
    The food uses shown in Table 2, using terminology from the cited
reference(s), were documented in articles that were published before or
during 1958; the cited references demonstrate that the use of these
enzyme preparations in a variety of foods was widely recognized by
1958. Therefore, the agency concludes that carbohydrase and protease
enzyme preparations derived from B. subtilis were in common use in food
prior to January 1, 1958.
2. Bacillus Amyloliquefaciens
    According to the petitioner (Refs. 8 and 14 through 16), the
species B. amyloliquefaciens was not classified under the name B.
amyloliquefaciens until it was taxonomically separated from the species
B. subtilis in the late 1980's (Refs. 17 and 18). Therefore, the
petitioner asserts, references in contemporaneous scientific literature
to pre-1958 food use of enzyme preparations from B. amyloliquefaciens
occur under the name B. subtilis.
    With respect to carbohydrase components of the petitioned enzyme

[[Page 19890]]

preparations, the petitioner cites scientific literature describing a
distinctive group of bacteria, within the group originally considered
to be B. subtilis, that are known to possess a high level of <greek-a>-
amylase activity and are currently designated as B. amyloliquefaciens
(Refs. 19 through 22). The petitioner also cites a scientific review
article (Ref. 23) that states that the source organism for commercial
preparations of <greek-a>-amylase from B. amyloliquefaciens was called
B. subtilis prior to its current designation as B. amyloliquefaciens.
With respect to the protease components of the petitioned enzyme
preparations, the petitioner cites a statement in the same scientific
review article (Ref. 23) that most bacterial protease preparations
produced before 1960 were derived from B. amyloliquefaciens.
    As FDA noted in the preamble to another final rule affirming an
enzyme preparation as GRAS (58 FR 27197 at 27199, May 7, 1993), the
taxonomic placement and name of an organism may change as a result of
scientific advances. If internationally accepted rules of nomenclature
are observed, references to a particular organism can be followed
historically in the scientific literature. Thus, changes in the
taxonomic placement of an organism should not affect the ability to
identify scientific references to the organism, including scientific
references to its toxigenicity, pathogenicity, or use in the production
of food or enzymes.In reviewing the petition, FDA has evaluated whether
the scientific information documenting pre-1958 food use of
bacterially-derived carbohydrase and protease enzyme preparations
pertains to carbohydrase and protease enzyme preparations from B.
amyloliquefaciens. Although it is not possible to determine
conclusively whether any one reference to B. subtilis in the scientific
literature refers to the species now referred to as B.
amyloliquefaciens, the totality of the scientific evidence supports a
determination that some carbohydrase and some protease enzyme
preparations that were described in scientific literature documenting
their common use in food before 1958 as derived from B. subtilis were
in fact derived from B. amyloliquefaciens. Therefore, the agency
concludes that carbohydrase and protease enzyme preparations derived
from B. amyloliquefaciens were in common use in food prior to January
1, 1958.

B. Corroborating Evidence of Safety

    Because enzymes are highly efficient catalysts, they are needed in
only minute quantities to perform their function. When used in
accordance with current good manufacturing practice (CGMP), the amounts
added to food represent only a minute fraction of the total food mass.
FDA estimates dietary exposure to enzyme preparations derived from B.
subtilis or B. amyloliquefaciens at 200 mg/person/day (Ref. 24). This
estimate is exaggerated because the agency used the total consumption
of microbially-derived enzyme preparations in food as an approximation
for the consumption of enzyme preparations derived from B. subtilis or
B. amyloliquefaciens. Thus, the estimate relies on the worst-case
assumption that all microbially-derived enzyme preparations that are
consumed in food are derived from B. subtilis or B. amyloliquefaciens.
This assumption is extremely conservative because there are numerous
microbially-derived enzyme preparations that are GRAS for use in food
(see, e.g., 21 CFR 184.1012, 184.1027, 184.1387, 184.1388, 184.1924,
and 184.1985).
1. The Enzyme Components
    Enzymes, including carbohydrase and protease enzymes in the enzyme
preparations that are the subject of this document, are naturally
occurring proteins that are ubiquitous in living organisms. A wide
variety of enzymes has always been present in human food. Many
naturally occurring enzymes remain active in unprocessed food and
therefore are consumed as active enzymes. For example, active enzymes
are present in fresh fruits and vegetables and are not inactivated
unless the fruits or vegetables are cooked (Refs. 1 and 25).
    Enzymes derived from microorganisms have been used as components of
foods that have been safely consumed as part of the diet throughout
human history (Ref. 26). For example, such common foods as bread and
yogurt are produced using enzymes derived from microorganisms (Refs. 26
and 27).
    The carbohydrase and protease enzymes in the enzyme preparations
that are the subject of this document are substantially equivalent \2\
to carbohydrase and protease enzymes from other microorganisms that FDA
has evaluated and found to be safe and that are routinely consumed as
part of a normal diet in the United States. For example, FDA has
affirmed the use of a mixed carbohydrase and protease enzyme
preparation derived from Bacillus licheniformis is GRAS (see 21 CFR
184.1027). In addition, carbohydrases derived from various fungi (e.g.,
Rhizopus niveus, Rhizopus oryzae, and A. niger) are approved for use as
secondary direct food additives (see 21 CFR 173.110, 173.130, and
173.120, respectively).
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    \2\ A 1996 report of the joint Food and Agriculture Organization
and World Health Organization (FAO/WHO) consultation group (Ref. 28)
stated that ``[s]ubstantial equivalence embodies the concept that if
a new food or food component is found to be substantially equivalent
to an existing food or food component, it can be treated in the same
manner with respect to safety (i.e. the food or food component can
be concluded to be as safe as the conventional food or food
component). Account should be taken of any processing that the food
or food component may undergo as well as the intended use and the
intake by the population.'' As discussed more fully in FDA's
proposal to amend the agency's regulations pertaining to substances
that are generally recognized as safe (62 FR 18938 at 18944, April
17, 1997), international expert groups such as the FAO/WHO
consultation group and the Organization for Economic Co-operation
and Development (OECD) consultation group have recommended that the
concept of ``substantial equivalence'' be applied to the safety
assessment of foods and substances intentionally added to food.
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    In general, issues relevant to a safety evaluation of proteins such
as the enzyme component of an enzyme preparation are potential toxicity
and allergenicity. Pariza and Foster (Ref. 1) note that very few toxic
agents have enzymatic properties, and those that do (e.g., diphtheria
toxin and certain enzymes in the venom of poisonous snakes) catalyze
unusual reactions that are not related to the types of catalysis that
are common in food processing and that are the subject of this
document. Further, as the agency has noted in the context of guidance
to industry regarding the safety assessment of new plant varieties,
enzymes do not generally raise safety concerns (57 FR 22984 at 23000,
May 29, 1992). Exceptions include enzymes that catalyze the formation
of toxic substances or substances that are not ordinarily digested and
metabolized. The catalytic activities of the enzymes that are the
subject of this document are well known; they split proteins or
carbohydrates into smaller subunits that are readily metabolized by the
human body and that do not have toxic properties.
    According to Pariza and Foster (Ref. 1), there have been no
confirmed reports of allergies or primary irritations in consumers
caused by enzymes used in food processing. There have been, however,
some reports of allergies and primary irritations from skin contact
with enzymes or inhalation of dust from concentrated enzymes (for
example, proteases used in the manufacture of laundry detergents)
(Refs. 29 through 31). These reports relate primarily to workers in
production plants (Ref. 30) and are not relevant to an evaluation of

[[Page 19891]]

the safety of ingestion of such enzymes in food.
    The 1977 report of the Select Committee on GRAS substances
concerning the plant enzyme papain (Ref. 29) supports the view that the
ingestion of an active protease at levels found in food products is not
likely to affect the human gastrointestinal tract, where many proteases
already exist at levels adequate to digest food:
    In common with other proteolytic enzymes, papain digests the
mucosa and musculature of tissues in contact with the active enzyme
for an appreciable period. Because there is no food use of papain
that could result in the enzyme preparation occurring in sufficient
amount in foods to produce these effects, this property does not
pose a dietary hazard.
    FDA concludes that generally available and accepted data and
information corroborate the safety of the enzyme components of the
enzyme preparations that are the subject of this document by
establishing that these enzyme components are identical or
substantially equivalent to enzymes that are known to have been safely
consumed in the diet for many years. FDA also concludes that generally
available and accepted data and information corroborate that the enzyme
components of the enzyme preparations that are the subject of this
document are nontoxigenic and nonallergenic when ingested.
2. Enzyme Sources, Manufacturing Methods, and Processing Aids
    Enzyme preparations used in food processing are usually not
chemically pure; in addition to the enzyme component(s), they may
contain other components derived from the production organism and the
fermentation medium, residual amounts of processing aids, and
substances added as stabilizers, preservatives, or diluents. The agency
has concluded that the enzyme components of the carbohydrase and
protease enzyme preparations derived from B. subtilis or B.
amyloliquefaciens do not raise safety concerns; therefore, the
remaining safety issue is whether other components of the enzyme
preparations are toxic or raise other safety concerns.
    a. Antibiotics. Some microorganisms are capable of producing
antibiotics, which are a special class of metabolites that can inhibit
the growth of, or kill, other microorganisms. Some microorganisms have
genetic traits that make them resistant to one or more antibiotics such
as penicillin, tetracycline, and kanamycin. These traits or markers are
often located on plasmids (extrachromosomal pieces of deoxyribonucleic
acid (DNA) that are easily transferred to other microorganisms in the
environment (e.g., in the gastrointestinal tract). The presence of
antibiotics in the food supply would be expected to favor the growth of
microorganisms resistant to the antibiotic, and thus could accelerate
the spread of antibiotic resistance among microorganisms, including
human pathogens, rendering them resistant to therapy with antibiotic
drugs. Therefore, experts have recommended that microbial-derived
enzyme preparations that are intended for food use not contain
clinically important antibiotics (Refs. 1 and 32).
    Accordingly, FDA has evaluated the potential for carbohydrase or
protease enzyme preparations derived from B. subtilis or B.
amyloliquefaciens to contain antibiotics as contaminants derived from
the bacterial source. Although Bacillus species are capable of
producing a number of linear or cyclic polypeptide antibiotics
following the exponential phase of growth as part of the process of
spore formation (Ref. 33), the production of antibiotics can be
repressed by selection of strains that produce low or undetectable
levels of antibiotics as well as by strict control of the growth
conditions. In addition, the enzyme preparations can be tested for the
presence of antibiotic activity by routine methods (Ref. 34) to ensure
that they do not contain antibiotics. Because of safety concerns about
the presence of antibiotics in substances added to food, a condition of
agency affirmation of GRAS status for the enzyme preparations that are
the subject of this document is that the enzyme preparations not
contain antibiotics.
    b. Toxicity and pathogenicity. A published scientific review
article (Ref. 23) states that Bacillus species, with the exception of
the B. cereus group (which does not include B. subtilis or B.
amyloliquefaciens) do not produce toxins. Another published scientific
review article on the safety of B. subtilis and B. amyloliquefaciens
(Ref. 35) notes that B. subtilis is consumed in large quantities in the
Japanese food natto. Further, according to a monograph on microbial
enzymes that was prepared under the auspices of the agency-initiated
review of GRAS substances conducted during the 1970's, there had been
no reported problems of pathogenicity or toxicity with enzyme
preparations derived from B. subtilis for use in food as of the time of
that review (Ref. 12).
    More recently, de Boer and Diderichsen (Ref. 35) searched the
scientific literature for references that might implicate B. subtilis
or B. amyloliquefaciens as a cause of human disease. These authors
characterized B. subtilis as an opportunistic microorganism with no
pathogenic potential to humans. Although they reported that cultures
from some patients with opportunistic infections have revealed the
presence of B. subtilis along with other microorganisms, they
attributed the presence of B. subtilis in these cultures to the virtual
ubiquity of this microorganism in the environment (e.g., B. subtilis
commonly occurs in the soil and can be isolated in the home environment
from sites such as the kitchen and bathroom). De Boer and Diderichsen
also noted that only patients treated with immunosuppressive drugs
appeared to be susceptible to such infections. Moreover, viable cells,
which are not present in finished enzyme preparations, would be a
prerequisite for any opportunistic infection in an immunocompromised
patient. De Boer and Diderichsen also reported that their search for
references on B. amyloliquefaciens infections revealed no such cases.
As discussed in section IV.A.2 of this document, any references to B.
amyloliquefaciens prior to the late 1980's would be expected to occur
under the name B. subtilis.
    A few reports have implicated B. subtilis as a potential source of
food poisoning when present as a contaminant in food (Refs. 36 and 37).
However, a particular strain of virtually any microorganism may, under
certain circumstances, mutate to become an opportunistic pathogen.
Therefore, FDA considered these reports in the context of: (1) The
information summarized in the monograph on microbial enzymes (Ref. 12);
(2) the scientific review article describing Bacillus species other
than those in the B. cereus group as nontoxigenic (Ref. 23); (3) the
documented consumption of B. subtilis bacteria in the Japanese food
natto (Ref. 35); and (4) the characterization by de Boer and
Diderichsen of B. subtilis as an opportunistic microorganism with no
pathogenic potential to humans (Ref. 36). Based on this information,
FDA concludes that nontoxigenic and nonpathogenic strains of B.
subtilis are widely available and have been safely used in a variety of
food applications. Because an enzyme preparation derived from a
toxigenic or pathogenic source would not be GRAS, a condition of agency
affirmation of GRAS status for the enzyme preparations that are the
subject of this document is that the bacterial strains used as a source
of these enzyme preparations be nontoxigenic and nonpathogenic.
    c. Manufacturing methods and processing aids. Enzyme preparations

[[Page 19892]]

that are manufactured in accordance with CGMP using the methods
described in section III.B of this document meet the general
requirements and additional requirements in the monograph on enzyme
preparations in the Food Chemicals Codex, 4th ed. (Ref. 3). Such enzyme
preparations are produced using substances that are acceptable for use
in foods and under culture conditions that ensure a controlled
fermentation, thus preventing the introduction of extraneous
microorganisms that could be the source of toxic materials and other
toxic substances (Ref. 3).
    FDA concludes that generally available and accepted data and
information corroborate the safety of carbohydrase and protease enzyme
preparations derived from nontoxigenic and nonpathogenic strains of B.
subtilis or B. amyloliquefaciens and manufactured in accordance with
CGMP by establishing that any added substances or impurities derived
from the enzyme source or introduced during the manufacturing of such
enzyme preparations would not be expected to present health concerns.

V. Comments

    FDA received seven comments in response to the filing notice and
none in response to the amendment notices. Of these, FDA received two
comments from food manufacturers, two from trade associations, one from
a manufacturer of enzymes for use in animal feed, one from a
pharmaceutical manufacturer, and one from a consumer group. Six
comments supported the petition for GRAS affirmation, stating that the
enzyme preparations included in the petition have a long history of use
in foods such as cheese, bread, and corn syrup.
    One comment stated that B. subtilis has a history of use in animal
feed and requested GRAS affirmation for this use. However, the petition
is for the use of certain enzyme preparations in human food, and not in
animal feed. Therefore, the agency finds that this comment is not
relevant to the petition.
    One comment asserted that enzyme preparations should not be
considered GRAS. The comment further asserted that the use of enzyme
preparations should be declared on the label of foods and that
consumers should be warned about hazards inherent in their use. The
comment stated that enzyme preparations are rarely purified to any
significant degree and contain a variety of cellular constituents and
metabolic debris. The comment further argued that, although enzyme
preparations are used at low levels and are inactivated after the
treatment of food, they may elicit allergic reactions and other
biological activities which could be detrimental to human health. In
support of this statement, the comment cited a published scientific
article (Ref. 38) that reported that enzyme preparations from B.
subtilis caused temporary weight loss and aggravated infection in mice
when injected into the abdominal cavity and caused hemolysis and
hemagglutination of sheep erythrocytes in in vitro studies.
    FDA has evaluated the comment and the article it cited. For the
following two reasons, FDA concludes that the study cited by the
comment is not relevant to food uses of the bacterial enzyme
preparations that are the subject of this document.
    First, the paper did not identify the composition of the B.
subtilis enzyme preparations tested. The preparations were intended for
use in laundry detergents; such nonfood grade enzyme preparations need
not conform to specifications for enzyme preparations used in food
processing. For example, nonfood grade enzyme preparations may include
processing aids that are not acceptable for food use. Because of such
differences, the results from the testing of laundry cleaning enzyme
preparations have little value in the safety assessment of food-
processing enzyme preparations.
    Second, in the cited study, adverse effects were observed in mice
after the intraperitoneal administration of B. subtilis autolysates.
However, exposure to enzyme preparations in food occurs by ingestion
and not by injection. The difference in the route of exposure is
particularly significant for assessing the significance of
immunological effects. With intraperitoneal administration, the
components of the immune system are directly exposed to a high level of
the test compound. This contrasts with exposure to enzyme preparations
in food, whereby low levels of the enzyme preparations are ingested and
undergo hydrolysis by digestive enzymes before any interaction with the
immune system. Pariza and Foster (Ref. 1) note that there are no
confirmed reports of allergic reactions in consumers caused by enzymes
used in food processing.
    Moreover, a report of the Joint Food and Agriculture Organization/
World Health Organization Expert Committee on Food Additives (JECFA)
corroborates the safety of food uses of enzyme preparations from B.
subtilis (Ref. 39). This report concluded that results from a 90-day
feeding study in rats showed no adverse effects. The test diet was meat
protein-based and supplemented with a protease enzyme preparation from
B. subtilis at a 1-percent level (equivalent to approximately 1 gram of
enzyme preparation per kilogram of body weight per day). This level is
more than 300 times greater than the highest level that would be
expected in the human diet (200 mg/person/day, or 3.3 mg/kg body weight
per day for a 60 kg person), as estimated in section IV.B of this
document.
    With respect to the comment's assertion that enzyme preparations
should be declared on the label of foods in which they are used, the
agency notes that under certain circumstances, applicable regulations
already require use of an enzyme preparation in a food to be declared
on the label, depending upon the nature of the enzyme preparation's use
and technical effect in the food. Section 403(i)(2) of the Federal
Food, Drug, and Cosmetic Act (21 U.S.C. 343(i)(2)) requires that all
ingredients of multi-ingredient foods be listed on the label of the
food. By regulation, FDA has exempted certain ingredients that are used
only as processing aids from this requirement. Section
101.100(a)(3)(ii)(a) and (a)(3)(ii)(c) (21 CFR 101.100(a)(3)(ii)(a) and
(a)(3)(ii)(c)) provides an exemption from the ingredient listing
requirement for processing aids that are added to a food for their
technical or functional effect during processing, but are either
removed from the food before packaging or are present in the finished
food at insignificant levels and do not have any technical or
functional effect in the finished food. Although many enzyme
preparations are used as processing aids in food (e.g., amylase
preparations used in the manufacture of glucose syrup and protease
preparations used in the manufacture of protein hydrolysates), other
enzyme preparations that are added during processing (e.g., protease
preparations used in tenderizing meat) are not processing aids as
defined in Sec. 101.100(a)(3)(ii) because they remain active in the
finished food product. For example, enzymes used in the manufacture of
swiss and cheddar cheese remain active in the finished cheese,
enhancing body, flavor, and aroma (49 FR 29242, July 19, 1984). Because
such effects in the finished food remove the enzymes from the
ingredient listing exemption for processing aids in
Sec. 101.100(a)(3)(ii)(c), the use of such enzymes must be declared on
the label. Therefore, whether a label declaration is needed for the use
of an enzyme preparation in a food will depend upon its function and
effect in the food.

[[Page 19893]]

VI. Conclusions

    The petitioner has provided generally available evidence
demonstrating that carbohydrase and protease enzyme preparations from
B. subtilis were in common use in food prior to 1958. FDA has
determined, under Sec. 170.30(a) and (c)(1), that this information
provides an adequate basis upon which to conclude that the safety of
these enzyme preparations for use in food is generally recognized among
the community of experts qualified by scientific training and
experience to evaluate the safety of food ingredients.
    The petitioner has also provided generally available evidence
demonstrating that the bacterium now known as B. amyloliquefaciens was
formerly included within the B. subtilis classification. Based on its
analysis of the data submitted, the agency concludes that the evidence
of common use in food pertains to carbohydrase and protease enzyme
preparations from the bacterium now known as B. amyloliquefaciens as
well as to carbohydrase and protease enzyme preparations from B.
subtilis.
    This evidence of common use in food prior to 1958 is corroborated
by information that the enzymes themselves and the sources from which
they are derived are nontoxic and nontoxicogenic, and that
manufacturing will not introduce impurities that would adversely affect
the safety of the finished enzyme preparations. Moreover, the
carbohydrase and protease enzyme preparations from B. subtilis and B.
amyloliquefaciens are substantially equivalent to enzymes naturally
present in foods that have been safely consumed in the human diet for
many years.
    Having evaluated the information in the petition, along with other
available information related to the use of these enzyme preparations,
the agency concludes that carbohydrase enzyme preparation and protease
enzyme preparation derived from either B. subtilis or B.
amyloliquefaciens are GRAS under conditions of use consistent with
CGMP. The agency is basing its conclusion on evidence of a substantial
history of safe consumption of the enzyme preparations in food by a
significant number of consumers prior to 1958, corroborated by the
other evidence summarized in section IV.B of this document.
    FDA is affirming that the use of these bacterially-derived
carbohydrase and protease enzyme preparations in food is GRAS with no
limits other than CGMP (21 CFR 184.1(b)(1)). To clarify the identity of
each enzyme preparation, the agency is including in Secs. 184.1148(a)
and 184.1150(a) the EC numbers of the enzymes that supply the
characterizing enzyme activities of each preparation. In order to make
clear that the affirmation of the GRAS status of these enzyme
preparations is based on the evaluation of specific uses, the agency is
including in Secs. 184.1148(c) and 184.1150(c) the technical effect and
the specific substances on which each enzyme preparation acts, although
the data show no basis for a potential risk from any foreseeable use of
these enzyme preparations.
    For simplicity, FDA is affirming the GRAS status of both
carbohydrase enzyme preparations in a single combined regulation that
describes the source of the enzyme as B. subtilis or B.
amyloliquefaciens, rather than affirming the GRAS status of
carbohydrase derived from B. subtilis separately from that of
carbohydrase derived from B. amyloliquefaciens. Likewise, FDA is
affirming the GRAS status of both protease enzyme preparations in a
single combined regulation that describes the source of the enzyme as
B. subtilis or B. amyloliquefaciens.
    To ensure that the enzyme preparations are of suitable purity for
use in food, FDA is including in the regulations the general
requirements and additional requirements for enzyme preparations in the
monograph ``Enzyme Preparations'' in the Food Chemicals Codex, 4th ed.
(1996) as general specifications for these enzyme preparations.
Furthermore, to ensure that the use of these enzyme preparations does
not promote the development of antibiotic resistance, the agency is
specifying that the enzyme preparations must be free of antibiotic
activity as determined by a suitable method (e.g., the method described
in Ref. 34).

VII. Environmental Considerations

    The agency has determined under 21 CFR 25.32(f) that this action is
of a type that does not individually or cumulatively have a significant
effect on the human environment. Therefore, neither an environmental
assessment nor an environmental impact statement is required.

VIII. Analysis for Executive Order 12866

    FDA has examined the impacts of this final rule under Executive
Order 12866. Executive Order 12866 directs Federal agencies to assess
the costs and benefits of available regulatory alternatives and, when
regulation is necessary, to select regulatory approaches that maximize
net benefits (including potential economic, environmental, public
health and safety effects; distributive impacts; and equity). According
to Executive Order 12866, a regulatory action is significant if it
meets any one of a number of specified conditions, including having an
annual effect on the economy of $100 million, adversely affecting in a
material way a sector of the economy, competition, or jobs, or raising
novel legal or policy issues. FDA finds that this final rule is not a
significant regulatory action as defined by Executive Order 12866. In
addition, the agency has determined that this final rule is not a major
rule for the purpose of Congressional review.
    The primary benefit of this action is to remove uncertainty about
the regulatory status of the petitioned substances. No compliance costs
are associated with this final rule because no new activity is required
and no current or future activity is prohibited by this rule.

IX. Regulatory Flexibility Analysis

    FDA has examined the impacts of this final rule under the
Regulatory Flexibility Act. The Regulatory Flexibility Act (5 U.S.C.
601-612) requires agencies to consider alternatives that would minimize
the economic impact of their regulations on small entities. No
compliance costs are associated with this final rule because no new
activity is required and no current or future activity is prohibited.
Accordingly, under the Regulatory Flexibility Act (5 U.S.C. 605(b)),
the agency certifies that this final rule will not have a significant
economic impact on a substantial number of small entities.

X. Paperwork Reduction Act of 1995

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

XI. Effective Date

    As this rule recognizes an exemption from the food additive
definition in the Federal Food, Drug, and Cosmetic Act, and from the
approval requirements applicable to food additives, no delay in
effective date is required by the Administrative Procedure Act, 5
U.S.C. 553(d). The rule will therefore be effective immediately (5
U.S.C. 553(d)(1)).

XII. References

    The following references have been placed on display in the Dockets
Management Branch (address above) and may be seen by interested persons

[[Page 19894]]

between 9 a.m. and 4 p.m., Monday through Friday.
    1. Pariza, M. W., and E. M. Foster, ``Determining the Safety of
Enzymes Used in Food Processing,'' Journal of Food Protection, vol.
46, pp. 453-468, 1983.
    2. Scott, D., ``Enzymes, Industrial,'' Encyclopedia of Chemical
Technology, edited by H. F. Mark et al., John Wiley and Sons, New
York, 3d ed., vol. 9, pp. 173-224, 1978.
    3. Monograph on ``Enzyme Preparations,'' Food Chemicals Codex,
National Academy Press, Washington, DC, 4th ed., pp. 131 and 133-
134, 1996.
    4. Underkofler, L. A., and W. J. Ferracone, ``Commercial
Enzymes--Potent Catalyzers that Promote Quality,'' Food Engineering,
vol. 29, pp. 123, 125-126, 130, and 133, 1957.
    5. Underkofler, L. A., R. R. Barton, and S. S. Rennet,
``Microbiological Process Report--Production of Microbial Enzymes
and Their Applications,'' Applied Microbiology, vol. 6, pp. 212-221,
1958.
    6. Beckhorn, E. J., M. D. Labee, and L. A. Underkofler,
``Production and Use of Microbial Enzymes for Food Processing,''
Journal of Agricultural and Food Chemistry, vol. 13, pp. 30-34,
1965.
    7. Comments of Ad Hoc Enzyme Technical Committee regarding FDA's
draft final regulations, entitled ``Enzymes Proposed for Affirmation
as GRAS,'' with a letter dated December 21, 1984, from Roger D.
Middlekauff, Ad Hoc Enzyme Technical Committee, to Kenneth A. Falci,
FDA.
    8. Response of the Enzyme Technical Association to the letter
dated December 13, 1985, of Lawrence Lin regarding GRASP 3G0016,
received February 18, 1986.
    9. Smythe, C. V., ``Microbiological Production of Enzymes and
Their Practical Applications,'' Economic Botany, vol. 5, pp. 126-
144, 1951.
    10. Reed, G., ``Enzymes, Industrial,'' Encyclopedia of Chemical
Technology, edited by R. E. Kirk and D. F. Othmer, Interscience
Encyclopedia, Inc., New York, 1st supplemental vol., pp. 294-312,
1957.
    11. Fogarty, W. M., editor, Microbial Enzymes and Biotechnology,
Applied Science Publishers, New York, NY, pp. 1-11, 22-25, 34-35,
111-113, 115-118, 162-173, 259-260, 282-286, 1983.
    12. Rogers, R.W., ``Monograph on Microbial Enzymes,''
Informatics, Inc., vol. 1, pp. 1-16, 1977.
    13. Reed, G., ``Industrial Enzymes--Now Speed Natural
Processes,'' Food Engineering, vol. 24, pp. 105-109, 1952.
    14. Response of the Enzyme Technical Association to the letter
dated June 26, 1986, of Lawrence J. Lin regarding GRASP 3G0016,
received with a letter dated October 3, 1986, from Roger D.
Middlekauff of the Enzyme Technical Association, to Lawrence J. Lin,
FDA.
    15. Letter dated August 17, 1995, from Gary L. Yingling, Enzyme
Technical Association, to Alan M. Rulis, FDA.
    16. Letter dated April 16, 1996, from Alice J. Caddow, Enzyme
Technical Association, to Linda Kahl, FDA.
    17. Priest, F. G., M. Goodfellow, L. A. Shute, and R. C. W.
Berkeley, ``Bacillus amyloliquefaciens sp. nov., nom. rev.,''
International Journal of Systematic Bacteriology, vol. 37, pp. 69-
71, 1987.
    18. Priest, F. G., M. Goodfellow, and C. Todd, ``A numerical
classification of the genus Bacillus,'' Journal of General
Microbiology, vol. 134, pp. 1847-1882, 1988.
    19. Baptist, J. N., M. Mandel, and R. J. Gherna, ``Comparative
zone electrophoresis of enzymes in the genus Bacillus,''
International Journal of Systematic Bacteriology, vol. 28, pp. 229-
244, 1978.
    20. Gordon, R. E., W. C. Haynes, and C. Hor-Nay Pang, ``The
genus Bacillus,'' U.S. Department of Agriculture, 1973.
    21. Welker, N. E., and L. L. Campbell, ``Unrelatedness of
Bacillus amyloliquefaciens and Bacillus subtilis,'' Journal of
Bacteriology, vol. 94, pp. 1124-1130, 1967.
    22. Welker, N. E., and L. L. Campbell, ``Comparison of the
alpha-amylase of Bacillus amyloliquefaciens and Bacillus subtilis,''
Journal of Bacteriology, vol. 94, pp. 1131-1135, 1967.
    23. Aunstrup, K., ``Production, Isolation, and Economics of
Extracellular Enzymes,'' Applied Biochemistry and Bioengineering,
vol. 2, pp. 27-69, 1979.
    24. Memorandum dated June 6, 1985, from John Modderman, Food
Additive Chemistry Evaluation Branch, to L. Lin, GRAS Review Branch.
``Enzymes proposed for GRAS affirmation based on history of use.''
    25. De Becze, G. I., ``Food Enzymes,'' Critical Reviews in Food
Technology, vol. 1, pp. 479-518, 1970.
    26. Phaff, H. J., M. W. Miller, and E. M. Mrak, ``The Life of
Yeasts, Their Nature, Activity, Ecology, and Relation to Mankind,''
Harvard University Press, Cambridge, MA, pp. 1-6, 133-149, 1966.
    27. Wilcox, G. ``Manufacture of Yogurt'' from ``Eggs Cheese and
Yogurt Processing,'' Noyes Data Corp., p. 269, 1971.
    28. ``Biotechnology and Food Safety,'' Food and Agriculture
Organization, Rome, 1996.
    29. ``Evaluation of the Health Aspects of Papain as a Food
Ingredient,'' Select Committee on GRAS Substances, Washington, DC,
available through U.S. Department of Commerce, National Technical
Information Service, Order No. PB-274-174, 1977.
    30. Fulwiler, R. D., ``Detergent Enzymes--An Industrial Hygiene
Challenge,'' American Industrial Hygiene Association Journal, vol.
32, pp. 73-81, 1971.
    31. ``Enzyme-containing Laundering Compounds and Consumer
Health,'' National Research Council/National Academy of Sciences,
National Technical Information Service, Washington, DC, Order No.
PB-204-118, 1971.
    32. Reanney, D., ``Extrachromosomal Elements as Possible Agents
of Adaptation and Development,'' Bacteriological Reviews, vol. 40,
pp. 552-590, 1976.
    33. Fogarty, W. M., P. J. Griffine, and A. M. Joyce, ``Enzymes
of Bacillus species--Part 1,'' Process Biochemistry, vol. 9, pp. 11-
13, 15, 17-18, 24, 1974.
    34. ``Determination of Antibiotic Activity,'' Compendium of Food
Additive Specifications, vol. 2, Joint FAO/WHO Expert Committee on
Food Additives (JECFA), Food and Agriculture Organization of the
United Nations, Rome, 1992.
    35. De Boer, A. S., and B. Diderichsen, ``On the Safety of
Bacillus subtilis and Bacillus amyloliquefaciens: A Review,''
Applied Microbiology and Biotechnology, vol. 36, pp. 1-4, 1991.
    36. Sneath, P. H. A., ``Endospore-forming Gram-Positive Rods and
Cocci,'' in Bergey's Manual of Systematic Bacteriology, edited by P.
H. A. Sneath et al., Williams & Wilkins, vol. 2, pp. 1104-1139,
Baltimore, 1986.
    37. Gilbert, R. J, P. C. B. Turnbull, J. M. Parry, and J. M.
Kramer, ``Bacillus cereus and other Bacillus Species: Their Part in
Food Poisoning and other Clinical Infections,'' in The Endospore-
forming Bacteria, edited by Berkeley and Goodfellow, Academic Press,
London, pp. 297-314, 1981.
    38. Dubos, R., ``Toxic Factors in Enzymes Used in Laundry
Products,'' Science, vol. 173, pp. 259-260, 1971.
    39. ``Toxicological Evaluation of Some Enzymes, Modified
Starches, and Certain Other Substances,'' 15th report of the Joint
Food and Agriculture Organization (FAO)/World Health Organization
(WHO) Expert Committee on Food Additives, WHO Technical Report
Series, FAO Nutrition Meetings Report Series, pp. 3-10, 1972.

List of Subjects in 21 CFR Part 184

    Food additives, Food ingredients, Incorporation by reference.
    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 184 is amended as follows:

PART 184--DIRECT FOOD SUBSTANCES AFFIRMED AS GENERALLY RECOGNIZED
AS SAFE

    1. The authority citation for 21 CFR part 184 continues to read as
follows:

    Authority: 21 U.S.C. 321, 342, 348, 371.

    2. Section 184.1148 is added to subpart B to read as follows:

Sec. 184.1148   Bacterially derived carbohydrase enzyme preparation.

    (a) Bacterially derived carbohydrase enzyme preparation is obtained
from the culture filtrate resulting from a pure culture fermentation of
a nonpathogenic and nontoxigenic strain of Bacillus subtilis or B.
amyloliquefaciens. The preparation is characterized by the presence of
the enzymes <greek-a>-amylase (EC 3.2.1.1) and <greek-b>-glucanase (EC
3.2.1.6), which catalyze the hydrolysis of O-glycosyl bonds in
carbohydrates.
    (b) The ingredient meets the general requirements and additional
requirements in the monograph on enzyme preparations in the Food
Chemicals Codex, 4th ed. (1996), pp. 128-135, which is incorporated by
reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies
are

[[Page 19895]]

available from the National Academy Press, 2101 Constitution Ave. NW.,
Washington, DC 20418, or may be examined at the Center for Food Safety
and Applied Nutrition's Library, 200 C St. SW., rm. 3321, Washington,
DC, or at the Office of the Federal Register, 800 North Capitol Street,
NW., Suite 700, Washington, DC. In addition, antibiotic activity is
absent in the enzyme preparation when determined by an appropriate
validated method such as the method ``Determination of antibiotic
activity'' in the Compendium of Food Additive Specifications, vol. 2,
Joint FAO/WHO Expert Committee on Food Additives (JECFA), Food and
Agriculture Organization of the United Nations, Rome, 1992. Copies are
available from Bernan Associates, 4611-F Assembly Dr., Lanham, MD
20706, or from The United Nations Bookshop, General Assembly Bldg., rm.
32, New York, NY 10017, or by inquiries sent to ``http://www.fao.org''.
Copies may be examined at the Center for Food Safety and Applied
Nutrition's Library, 200 C St. SW., rm. 3321, Washington, DC.
    (c) In accordance with Sec. 184.1(b)(1), the ingredient is used in
food with no limitation other than current good manufacturing practice.
The affirmation of this ingredient as GRAS as a direct food ingredient
is based upon the following current good manufacturing practice
conditions of use:
    (1) The ingredient is used as an enzyme as defined in
Sec. 170.3(o)(9) of this chapter to hydrolyze polysaccharides (e.g.,
starch).
    (2) The ingredient is used in food at levels not to exceed current
good manufacturing practice.
    3. Section 184.1150 is added to subpart B to read as follows:

Sec. 184.1150 Bacterially-derived protease enzyme preparation.

    (a) Bacterially derived protease enzyme preparation is obtained
from the culture filtrate resulting from a pure culture fermentation of
a nonpathogenic and nontoxigenic strain of Bacillus subtilis or B.
amyloliquefaciens. The preparation is characterized by the presence of
the enzymes subtilisin (EC 3.4.21.62) and neutral proteinase (EC
3.4.24.28), which catalyze the hydrolysis of peptide bonds in proteins.
    (b) The ingredient meets the general requirements and additional
requirements in the monograph on enzyme preparations in the Food
Chemicals Codex, 4th ed. (1996), pp. 128-135, which is incorporated by
reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies
are available from the National Academy Press, 2101 Constitution Ave.
NW., Washington, DC 20418, or may be examined at the Center for Food
Safety and Applied Nutrition's Library, 200 C St. SW., rm. 3321,
Washington, DC, or at the Office of the Federal Register, 800 North
Capitol Street, NW., Suite 700 Washington, DC. In addition, antibiotic
activity is absent in the enzyme preparation when determined by an
appropriate validated method such as the method ``Determination of
antibiotic activity'' in the Compendium of Food Additive
Specifications, vol. 2, Joint FAO/WHO Expert Committee on Food
Additives (JECFA), Food and Agriculture Organization of the United
Nations, Rome, 1992. Copies are available from Bernan Associates, 4611-
F Assembly Dr., Lanham, MD 20706, or from The United Nations Bookshop,
General Assembly Bldg., rm. 32, New York, NY 10017, or by inquiries
sent to ``http://www.fao.org''. Copies may be examined at the Center
for Food Safety and Applied Nutrition's Library, 200 C St. SW., rm.
3321, Washington, DC.
    (c) In accordance with Sec. 184.1(b)(1), the ingredient is used in
food with no limitation other than current good manufacturing practice.
The affirmation of this ingredient as GRAS as a direct food ingredient
is based upon the following current good manufacturing practice
conditions of use:
    (1) The ingredient is used as an enzyme as defined in
Sec. 170.3(o)(9) of this chapter to hydrolyze proteins or polypeptides.
    (2) The ingredient is used in food at levels not to exceed current
good manufacturing practice.

    Dated: March 26, 1999.
L. Robert Lake,
Director, Office of Policy, Planning and Strategic Initiatives, Center
for Food Safety and Applied Nutrition.
[FR Doc. 99-10011 Filed 4-22-99; 8:45 am]
BILLING CODE 4160-01-F