Federal Register, Volume 60 Issue 122 (Monday, June 26, 1995)

[Federal Register Volume 60, Number 122 (Monday, June 26, 1995)]
[Rules and Regulations]
[Pages 32904-32912]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 95-15239]

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DEPARTMENT OF HEALTH AND HUMAN SERVICES
21 CFR PART 184

[Docket No. 84G-0257]

Enzyme Preparations From Animal and Plant Sources; 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
certain enzyme preparations derived from animal and plant sources 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). The
following enzyme preparations derived from animal sources are affirmed
as GRAS in this final rule: Catalase (bovine liver), animal lipase,
pepsin, trypsin, and pancreatin (as a source of protease activity). The
following enzyme preparations derived from plant sources are affirmed
as GRAS in this final rule: Bromelain, ficin, and malt.

DATES: Effective June 26, 1995. 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 listed
in 21 CFR 184.1024(b), 184.1034(b), 184.1316(b), 184.1415(b),
184.1443a(b), 184.1583(b), 184.1595(b), and 184.1914(b), effective June
26, 1995.

FOR FURTHER INFORMATION CONTACT: Laura M. Tarantino, 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:

Table of Contents

I. Introduction
II. Standards for GRAS Affirmation
III. Background
A. Enzymes
B. Enzyme Nomenclature
C. Enzyme Preparations that are the Subject of this Document
1. Introduction
2. Animal-derived Enzyme Preparations
3. Plant-derived Enzyme Preparations
IV. Safety Evaluation
A. Pre-1958 History of Use in Food
B. Corroborating Evidence of Safety
1. The Enzyme Component
2. Enzyme Sources and Processing Aids
3. Dietary Exposure
V. Comments
VI. Conclusions
VII. Environmental Impact
VIII. Economic Impact
IX. 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: Aspergillus niger,
var. (lipase, catalase, glucose oxidase, and carbohydrase); Bacillus
subtilis, var. (carbohydrase and protease mixtures); Rhizopus oryzae
(carbohydrase); and Saccharomyces species (carbohydrase).
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, rm. 1-23, 12420 Parklawn Dr.,
Rockville, MD 20857. 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; and in the Federal Register
of June 23, 1987 (52 FR 23607), proposing affirmation that the enzyme
preparation protease from A. niger is 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, [[Page 32905]] 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). The agency
concludes that rennet, bovine rennet, and papain are already affirmed
as GRAS and listed in existing regulations and need not be addressed
further.
In letters to FDA (Refs. 1 and 2), the petitioner asserted that the
enzyme preparation malt (amylase) includes extracts from germinated
(malted) barley or ungerminated (unmalted) barley. In addition, certain
published references (Refs. 3 and 4) submitted by the petitioner
describe the enzyme preparation pancreatin as a substance containing
the enzymes amylase, lipase, and protease.
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.
This final rule is a partial response to the petition and addresses
only enzyme preparations from animal and plant sources. Microbial
enzyme preparations will be dealt with separately in a future issue of
the Federal Register. Furthermore, in accordance with the September 20,
1993, Federal Register notice, FDA's determination of the GRAS status
of the enzyme preparation malt includes only the enzyme preparation
derived from malted barley extracts. Likewise, FDA's determination of
the GRAS status of the enzyme preparation pancreatin includes only the
use of pancreatin as a protease.

II. Standards for GRAS Affirmation

Pursuant to 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 evidence required for approval of a food
additive regulation, and ordinarily is based upon generally available
data and information.
For the enzyme preparations from animal and plant sources that are
the subject of this document, the Enzyme Technical Association based
its request for affirmation of GRAS status on a history of safe food
use prior to 1958. In the preamble to a proposed rule amending
Sec. 170.30, which was published in the Federal Register of July 2,
1985 (50 FR 27294) (final rule published in the Federal Register of May
10, 1988 (53 FR 16544)), FDA stated that 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.

III. Background

A. Enzymes

Enzymes are proteins or conjugated proteins,^{1 produced by
plants, animals, and microorganisms, that function as biochemical
catalysts (Ref. 5). Further, 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. 6). The practical applications
of enzymes used in food processing include the conversion of starch to
sugars in brewing, the tenderizing of sausage casings and meat, and the
partial hydrolysis (breakdown) of proteins that would otherwise form a
haze when beer is chilled (Ref. 7).

\1\A conjugated protein is a protein that contains a nonamino
acid moiety such as a carbohydrate.
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B. Enzyme Nomenclature

Enzymes were originally known principally by their trivial (common
or historical) names. These trivial names typically were based on one
of two methods of nomenclature: (1) By the addition of ``-in'' or ``-
ain'' as a suffix to a root indicating the source of the enzyme (e.g.,
papain from papaya or pancreatin from pancreas); or (2) by the addition
of the suffix ``-ase'' to a root indicating the substrate (specific
reactant) for the enzyme (e.g., lactase, which acts on the substrate
lactose) (Ref. 8). Some proteases, however, have trivial names that are
not based on either of these two methods (e.g., trypsin).
In 1956, the Third International Congress of the International
Union of Biochemistry (IUB) organized a Commission on Enzymes to devise
a systematic strategy for naming enzymes. The system developed by the
Commission on Enzymes combined a naming system and a numbering system
(Ref. 8). With the exception of most proteases, the systematic name is
derived from the names of the substrate, product, and type of
reaction.^{2 The systematic number is based on the class and
subclasses to which the enzyme belongs. The two classes of enzymes in
the numbering system relevant to this document are class 1,
oxidoreductases (e.g., catalase), which are active in biological
oxidation and reduction; and class 3, hydrolases (e.g., glycosidases
(carbohydrases), lipases, and proteases), which catalyze the splitting
of chemical bonds by the addition of water.

\2\In general, proteolytic enzymes are not sufficiently defined
to apply short systematic names.
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The following examples illustrate the trivial name, functions, and
Enzyme Commission (EC) name and number of enzymes that are components
of some of the enzyme preparations that are the subject of this
document (Refs. 9 through 11).
-amylase. Hydrolysis of -1,4-glucan bonds in
polysaccharides (starch, glycogen, etc.), yielding dextrins and oligo-
and monosaccharides (1,4--D-glucan glucanohydrolase, EC
3.2.1.1).
Catalase. Decomposition of hydrogen peroxide (H2O2),
yielding water and molecular oxygen (H2O2:H2O2
oxidoreductase, EC 1.11.1.6).

C. Enzyme Preparations That Are the Subject of This Document

1. Introduction
The enzyme preparations that are the subject of this document are
derived from animal or plant sources. They contain one or more active
enzymes and may also contain diluents, preservatives, antioxidants, and
other substances. Table 1 includes characterizing enzyme
activities^{3 of the animal- and plant-derived enzyme preparations
that are the subject of this document, as well as their Chemical
Abstracts Service Registry Numbers (CAS Reg. Nos.) and EC numbers as
appropriate (Refs. 3, 4, and 9 through 11). [[Page 32906]]

\3\The activity of a commercial product is a measurement of the
rate of the reaction catalyzed by the enzyme of interest in the
enzyme preparation, and is usually expressed in activity units per
unit weight of the product (Ref. 8). The enzyme preparation is then
diluted or concentrated until the activity is within a certain
desired range.

Table 1.--Enzyme Activities, CAS Reg. Nos., and EC Numbers Associated
With Some Enzyme Preparations
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CAS Reg.
Enzyme preparation Enzyme activity No. EC No.
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Catalase................ Catalase............ 9001-05-2 1.11.1.6
Animal lipase........... Lipase.............. 9001-62-1 3.1.1.3
Pepsin.................. Protease............ 9001-75-6 3.4.23.1
Trypsin................. Protease............ 9002-07-7 3.4.21.4
Pancreatin\1\........... Protease............ 8049-47-6 N/A
Amylase
Lipase
Bromelain............... Protease............ 9001-00-7 3.4.22.32
Ficin................... Protease............ 9001-33-6 3.4.22.3
Malt\2\................. -amylase... N/A 3.2.1.1
-amylase... ........... 3.2.1.2
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\1\Pancreatin is identified by a CAS Reg. No. but does not have an EC
number.
\2\The -amylase and -amylase enzyme activities in malt
are identified by EC number, but malt does not have a CAS Reg. No.

2. Animal-Derived Enzyme Preparations
a. Sources. The animal-derived enzyme preparations that are the
subject of this document are derived from a variety of animal sources.
Catalase is obtained from bovine liver (Ref. 9). Animal lipase is
obtained from the edible forestomach tissue of calves, kids, or lambs,
or from animal pancreatic tissue (Ref. 9). Pepsin is obtained from the
glandular layer of hog stomach (Ref. 9). Trypsin is obtained from
porcine or bovine pancreas (Ref. 9). Pancreatin is also obtained from
porcine or bovine pancreas (Refs. 3 and 4). These source materials for
bovine liver catalase, animal lipase, pepsin, trypsin, and pancreatin
were described by Tauber in 1949 (Ref. 12) and by Reed, in Kirk and
Othmer in 1957 (Ref. 13).

b. Methods of manufacture. The animal-derived enzyme preparations
that are the subject of this document are produced either as tissue
preparations (powders) or aqueous extracts of tissues from edible
animals (Refs. 8, 9, 12, and 13). In the tissue preparation method, the
animal tissue is ground with processing aids, such as sodium chloride
and skim milk powder. In the aqueous extract method, the enzyme
preparation may remain in aqueous solution, or it can be precipitated
by adding a solvent such as acetone or methyl alcohol. For example,
pepsin can be prepared by the aqueous extraction of animal tissue,
while animal lipase can be prepared by the tissue preparation method as
well as the aqueous extraction method.

c. Technical effects. Pre-1958 uses in food of animal-derived
enzyme preparations are listed in Table 2, using terminology from the
cited reference(s) published before or during 1958.

Table 2.--Applications of Animal-Derived Enzymes in Food Prior to 1958
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Technical effect or
Enzyme preparation Enzyme activity Food categories industry application References
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Pepsin............... Protease............. Beer................. Chillproofing........ 7, 13, 14, 15
Condiments........... Not reported......... 15
Evaporated milk...... Stabilization........ 15
Pancreatin........... Protease............. Milk................. Prevention of 13, 15
oxidation flavor.
Milk................. Protein hydrolysis... 13, 15
Evaporated milk...... Stabilization........ 15
Trypsin.............. Protease............. Milk................. Antioxidant.......... 16
Lipase............... Lipase............... Italian type cheeses. Flavor production.... 13, 17, 18
Catalase............. Catalase............. Milk................. Removal of peroxide 13, 15
after sterilization.
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3. Plant-Derived Enzyme Preparations

a. Sources. Bromelain is obtained from the pineapples Ananas
comosus and A. bracteatus L. (Ref. 9). Ficin is obtained from the latex
of species of the genus Ficus (fig tree) (Ref. 9). Malt is obtained
from barley after controlled germination (Ref. 19). These source
materials for bromelain, ficin, and malt were described by Tauber in
1949 (Ref. 12) and by Reed in 1957 (Ref. 13). [[Page 32907]]
b. Methods of manufacture. Bromelain is obtained from pineapple
juice (pressed from the stems of pineapples that remain after
harvesting the fruit) by precipitation with alcohol or ammonium sulfate
(Refs. 8, 12, and 13). Ficin is obtained from the latex of a variety of
tropical fig trees by precipitation with acetone or alcohol (Refs. 9,
12, and 14).
Malt is produced from germinated barley. The petition describes the
following process for the manufacture of malt (Ref. 19). Barley is
softened by a series of steeping operations in water at 10 deg.C to 30
deg.C until the moisture content of the kernels reaches 40 to 50
percent. The grain is then germinated under controlled conditions for a
period of up to 7 days. Reducing substances are added to activate the
enzymes. Solids are removed from the extract, which is concentrated,
stabilized, and standardized. The resultant syrup is usually a brown,
sweet, and viscous liquid with a specific gravity of approximately 1.1
to 1.3 at 25 deg.C.
c. Technical effects. Pre-1958 uses in food of plant-derived enzyme
preparations are listed in Table 3, using terminology from the cited
reference(s) published before or during 1958.

Table 3.--Applications of Plant-Derived Enzymes in Food Prior to 1958
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Technical effect or
Enzyme preparation Enzyme activity Food categories industry application References
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Malt................. Amylase.............. Bread................ Baking............... 7, 14, 15
Beer................. Mashing.............. 14, 15
Precooked baby Not reported......... 15
cereals.
Breakfast cereals.... Not reported......... 14, 15
Distilled beverages.. Mashing.............. 15
Bromelain............ Protease............. Beer................. Chillproofing........ 13, 14, 15
Condiments........... Not reported......... 15
Milk................. Protein hydrolysis... 15
Evaporated milk...... Stabilization........ 15
Meat................. Tenderizing, 13, 14, 15, 20
softening tissue.
Sausage casings...... Tenderizing.......... 14, 15
Fish................. Condensing fish 15
solubles.
Ficin................ Protease............. Meat................. Softening............ 20
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IV. Safety Evaluation

A. Pre-1958 History of Use in Food

Enzymes have been used for many years in the production and
processing of food, for example, in the baking, dairy, and brewing
industries (e.g., see Refs. 7, 13, and 14). The consumption of food
produced using these enzymes has produced no evidence of an associated
human health hazard.
The petitioner provided generally available information, including
published papers and review articles, showing that the animal- and
plant-derived enzyme preparations that are the subject of this document
were commonly used in food prior to 1958. For example, the pre-1958
food uses shown in Tables 2 and 3 were documented in articles that were
published in or before 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 the enzyme
preparations that are the subject of this document were in common use
in food prior to January 1, 1958.

B. Corroborating Evidence of Safety

1. The Enzyme Components
A wide variety of enzymes has always been present in human food.
Moreover, many naturally occurring enzymes in the cells of animals and
plants used for food remain active after cell death. For example,
active enzymes are present in fresh fruits and vegetables and are not
inactivated unless the fruits or vegetables are cooked (Refs. 6 and
21).
The enzymes that are the subject of this document are naturally
occurring proteins that are ubiquitous in living organisms. They are
derived from animals and plants that have been used as sources of food,
and are identical or substantially similar^{4 to enzymes that have
been safely consumed as part of the diet throughout human history.

\4\Enzymes that have the same function and that are identified
by the same name and EC number often differ slightly in structure
and properties when they are obtained from different sources. For
example, the structure of an enzyme isolated from one tissue (such
as the liver) of one animal species, may differ slightly from that
of the same enzyme isolated from a different tissue from the same
species, or from the liver of another animal species. In part
because of this variability, the diet routinely contains many
thousands of different enzyme protein molecules. The concept of
substantial similarity relative to food safety assessment has
recently been discussed by several expert groups. For example, a
report prepared by an expert group of the Organization for Economic
Co-operation and Development (OECD) concluded, in part, ``[I]f 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. No additional safety concerns would
be expected.'' (``Safety Evaluation of Foods Derived by Modern
Biotechnology: Concepts and Principles,'' OECD, 1993, Paris).
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Issues relevant to a safety evaluation of proteins from food
sources are potential toxicity and allergenicity. Pariza and Foster
(Ref. 6) 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, the agency has recently noted,
in the context of guidance to industry regarding the safety assessment
of new plant varieties, that newly introduced enzymes do not generally
raise safety concerns (Ref. 22). Exceptions include enzymes that
produce substances that are not ordinarily digested and metabolized, or
that produce toxic substances. The functions of the enzymes that are
the subject of this document are well known; they split proteins,
carbohydrates, lipids, or other substances (e.g., hydrogen peroxide)
into smaller subunits that do not have toxic properties and that are
readily metabolized by the human body.
The agency is not aware of any reports of allergic reactions
associated with the ingestion in food of the enzymes that are the
subject of this document. 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. 23 through 25).
These reports relate primarily to workers in production plants (Ref.
24) and are not relevant to an evaluation of the safety of ingestion of
such enzymes in food. [[Page 32908]] Moreover, Pariza and Foster (Ref.
6) note that there are no confirmed reports of primary irritations in
consumers caused by enzymes used in food processing.
The 1977 report of the Select Committee on GRAS substances
concerning the plant enzyme papain (Ref. 23) 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.

In summary, the enzyme components of the preparations that are the
subject of this document are identical or substantially similar to
enzymes that are known to have been safely consumed in the diet; they
do not result in the production of toxic substances; and their use in
food for many years has not been associated with reports of
allergenicity or primary irritation. Therefore, the agency finds that
the presence of the enzyme components does not create a basis for
concern about the safety of the enzyme preparations.
2. Enzyme Sources and Processing Aids
The agency has concluded that the enzyme components of enzyme
preparations do not raise safety concerns; therefore, the relevant
safety issue becomes whether the enzyme preparations contain toxic
contaminants. Enzyme preparations used in food processing are usually
not chemically pure but contain, in addition to the enzyme component,
materials that derive from the enzyme source, as well as from the
manufacturing methods used to generate the finished enzyme preparation.
In accordance with Sec. 170.30(h)(1), the enzyme preparations
affirmed as GRAS in this document must comply with the general
requirements and additional requirements for enzyme preparations in the
Food Chemicals Codex, 3d ed. (Ref. 9). When the animal-derived enzyme
preparations that are the subject of this document are produced in
accordance with current good manufacturing practice (CGMP), they are
obtained from animal tissues that comply with applicable Federal meat
inspection requirements and that are handled in accordance with good
hygienic practices (Ref. 9). Similarly, when produced in accordance
with CGMP, the plant material used in the production of enzymes
consists of components that leave no residues harmful to health in the
finished food under normal conditions of use (Ref. 9).
The enzyme preparations may contain substances, such as salts,
preservatives, or stabilizers, that are used in their preparation and
purification. When used in accordance with CGMP, these processing aids
are substances that are acceptable for general use in foods (Ref. 9).
As always, any of these substances that are intended to become or
become functional components of the enzyme preparation must be GRAS
substances or food additives approved for use in the manufacture of
enzyme preparations. Therefore, the agency concludes that the presence
of added substances and impurities derived from the enzyme source or
introduced by manufacturing does not present a basis for concern about
the safety of the enzyme preparations.
3. Dietary Exposure
Because enzymes are highly efficient catalysts, they are needed in
only minute quantities to perform their function. When used in
accordance with CGMP, the amounts added to food represent only a minute
fraction of the total food mass. The history of common use in food for
many years of the enzyme preparations that are the subject of this
document has produced no evidence of an associated hazard; further,
there is no reason to believe that use of these enzyme preparations at
levels needed to perform their functions would raise a safety concern.
Therefore, the agency concludes that no limits other than CGMP are
needed to ensure safe use.

V. Comments

FDA received seven letters in response to the filing notice and
none in response to the amendment notices. Three comments concerned
microbially derived enzyme preparations, which will be addressed in a
separate document. Of the remaining four comments, one came from a food
manufacturer, two from trade associations, and one from a consumer
group. Three comments supported the petition for GRAS affirmation of
the enzyme preparations included in the petition, stating that these
enzyme preparations have a long history of use in foods such as cheese,
bread, and corn syrup.
One comment asserted that enzyme preparations should not be
considered GRAS, and their use should be declared on the label of foods
to warn consumers 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. 26)
which 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. Because this article concerns
microbially derived enzyme preparations injected directly into the
abdominal cavity, it is not relevant to this rulemaking, which concerns
animal- and plant-derived enzyme preparations consumed by mouth.
The agency also 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. These regulatory
requirements are discussed below.
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. Sections 101.100(a)(3)(ii)(a) and (a)(3)(ii)(c) (21 CFR
101.100(a)(3)(ii)(a) and (a)(3)(ii)(c)) provide 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., the use of
amylase preparations in the manufacture of glucose syrup and the use of
protease preparations in the manufacture of protein hydrolyzates),
other enzyme preparations are not used solely as processing aids in the
manufacture of foods (e.g., the use of lipase preparations for flavor
production in cheeses and the use of protease preparations in
tenderizing meat). In these cases, the enzymes remain active
[[Page 32909]] in the finished food product, functioning as an integral
part of the food by 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 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.

VI. Conclusions

The petitioner has provided generally available evidence
demonstrating that the enzyme preparations under consideration were in
common use in food prior to 1958. As provided for under Sec. 170.30(a)
and (c)(1), FDA has determined that this information provides an
adequate basis upon which to conclude that the use of these enzyme
preparations in food is generally recognized as safe among the
community of experts qualified by scientific training and experience to
evaluate the safety of food ingredients.
This evidence of common use in food prior to 1958 without any
reported adverse effects from consumption is corroborated by the
absence of any reports of toxicity resulting from use of the enzyme
preparations in food since 1958, by information that the enzymes
themselves and the sources from which they are derived are nontoxic,
and by evidence that manufacturing will not introduce impurities that
will adversely affect the safety of the finished enzyme preparations.
Moreover, the enzyme preparations that are the subject of this document
are substantially similar to enzymes naturally present in foods that
have been safely consumed in the human diet for centuries.
Having evaluated the information in the petition, along with other
available information that related to the use of these enzyme
preparations, the agency concludes that the following enzyme
preparations derived from animal or plant sources are GRAS under
conditions of use consistent with CGMP: Bromelain, catalase (bovine
liver), ficin, animal lipase, malt, pancreatin (as a source of protease
activity), pepsin, and trypsin. 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 above.
FDA is therefore affirming that the use of the enzyme preparations
that are the subject of this document is GRAS with no limits other than
CGMP (21 CFR 184.1(b)(1)). The agency further concludes that the
general and additional requirements for enzyme preparations in the Food
Chemicals Codex, 3d ed. (1981), pp. 107-110, are adequate as minimum
criteria for food-grade preparations of these enzymes.
To clarify the identity of each enzyme preparation, the agency is
including in Secs. 184.1024(a), 184.1034(a), 184.1316(a), 184.1415(a),
184.1443a(a), 184.1583(a), 184.1595(a), and 184.1914(a), the EC
number(s) of the enzyme preparation or of the characterizing enzyme
activity(ies) for food use of the preparation^{5. 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. 184.1024(c), 184.1034(c), 184.1316(c), 184.1415(c),
184.1443a(c), 184.1583(c), 184.1595(c), and 184.1914(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.

\5\The EC number is sufficient to define the characterizing
activity in the enzyme preparation. Therefore, FDA is not including
the EC systematic name in the regulation.
---------------------------------------------------------------------------

VII. Environmental Impact

The agency has determined under 21 CFR 25.24(b)(7) 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. Economic Impact

FDA has examined the impact of this final rule affirming the GRAS
status of enzyme preparations from animal and plant sources under
Executive Order 12866 and the Regulatory Flexibility Act (Pub. L. 96-
354). 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). The
Regulatory Flexibility Act requires Federal agencies to minimize the
economic impact of their regulations on small businesses.
The agency finds that this final rule is not a significant
regulatory action as defined by Executive Order 12866. The rule
requires no change in current industry practice concerning the
manufacture and use of these substances. Compliance costs to firms are
therefore estimated to be zero. The substances that are the subject of
this document pose no health risks to consumers when used as intended.
Costs to consumers are therefore also estimated to be zero.
In accordance with the Regulatory Flexibility Act, FDA also has
determined that this final rule will not have a significant adverse
impact on a substantial number of small businesses.

IX. 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. 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.
2. Letter dated September 20, 1985, from Roger D. Middlekauff,
Enzyme Technical Association, to Lawrence J. Lin, FDA.
3. Monograph on ``Pancreatin,'' U.S. Pharmacopeia, 21st revision,
the United States Pharmacopeial Convention, Inc., Rockville, MD, pp.
777-778, 1985.
4. Monograph on ``Pancreatin,'' U.S. Pharmacopeia, 6th supp., the
United States Pharmacopeial Convention, Inc., Rockville, MD, pp.
2595-2597, 1987.
5. Morris, W., editor, The American Heritage Dictionary of the
English Language, Houghton Mifflin Co., Boston, MA, p. 438, 1976.
6. Pariza, M. W., and E. M. Foster, ``Determining the Safety of
Enzymes Used in Food Processing,'' Journal of Food Protection,
46:453-468, 1983.
7. Reed, G., ``Industrial Enzymes--Now Speed Natural Processes,''
Food Engineering, 24:105-109, 1952.
8. Scott, D., ``Enzymes, Industrial,'' Encyclopedia of Chemical
Technology, Mark, H. F. et al., editors, John Wiley and Sons, New
York, 3d ed., 9:173-224, 1978.
9. Monograph on ``Enzyme Preparations,'' Food Chemicals Codex,
National Academy Press, Washington, DC, 3d ed., pp. 107-110, and
480-481, 1981.
10. IUB, ``Enzyme Nomenclature 1992,'' Academic Press, New York, pp.
116, 307, 346, 388, 399, 402-403, 1992.
11. IUB, ``Enzyme Nomenclature 1964,'' Academic Press, New York, pp.
66-67, 86-87, 126-131, 136-149, and 170-171, 1965.
12. Tauber, H., ``The Chemistry and Technology of Enzymes,'' John
Wiley and Sons, New York, pp. 25-26, 130-131, 140, 145-151, 163-167,
192-193, and 327-335, 1949. [[Page 32910]]
13. Reed, G., ``Enzymes, Industrial,'' Encyclopedia of Chemical
Technology, Kirk, R. E. and D. F. Othmer, editors, Interscience
Encyclopedia, Inc., New York, 1st supplemental vol., pp. 294-312,
1957.
14. Underkofler, L. A., and W. J. Ferracone, ``Commercial Enzymes--
Potent Catalyzers that Promote Quality,'' Food Engineering, 29:123,
125-126, 130, and 133, 1957.
15. Underkofler, L. A., R. R. Barton, and S. S. Rennet,
``Microbiological Process Report--Production of Microbial Enzymes
and Their Applications,'' Applied Microbiology, 6:212-221, 1958.
16. Smythe, C. V., ``Microbiological Production of Enzymes and Their
Practical Applications,'' Economic Botany, 5:126-144, 1951.
17. Harper, W. J. and J. E. Long, ``Italian Cheese Ripening. IV.
Various Free Amino and Fatty Acids in Commercial Provolone Cheese,''
Journal of Dairy Science, 39:129-137, 1956.
18. Long, J. E., and W. J. Harper, ``Italian Cheese Ripening. VI.
Effects of Different Types of Lipolytic Enzyme Preparations on the
Accumulation of Various Free Fatty and Free Amino Acids and the
Development of Flavor in Provolone and Romano Cheese,'' Journal of
Dairy Science, 39:245-252, 1956.
19. 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.
20. ``List of Chemicals Approved Under Meat Inspection Act Before
September 6, 1958, Which are Exempted from the 1958 Food Additives
Amendment of the Federal Food, Drug, and Cosmetic Act,'' Food Drug
Cosmetic Law Journal, 13:834-840, 1958.
21. De Becze, G. I., ``Food Enzymes,'' Critical Reviews in Food
Technology,'' 1:479-518, 1970.
22. FDA, ``Statement of Policy: Foods Derived from New Plant
Varieties,'' 57 FR 22984 at 23005; May 29, 1992.
23. ``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.
24. Fulwiler, R. D., ``Detergent Enzymes--An Industrial Hygiene
Challenge,'' American Industrial Hygiene Association Journal, 32:73-
81, 1971.
25. ``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.
26. Dubos, R., ``Toxic Factors in Enzymes Used in Laundry
Products,'' Science, 173:259-260, 1971.

List of Subjects in 21 CFR Part 184

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: Secs. 201, 402, 409, 701 of the Federal Food, Drug,
and Cosmetic Act (21 U.S.C. 321, 342, 348, 371).

2. Section 184.1024 is added to subpart B to read as follows:

Sec. 184.1024 Bromelain.

(a) Bromelain (CAS Reg. No. 9001-00-7) is an enzyme preparation
derived from the pineapples Ananas comosus and A. bracteatus L. It is a
white to light tan amorphous powder. Its characterizing enzyme activity
is that of a peptide hydrolase (EC 3.4.22.32).
(b) The ingredient meets the general requirements and additional
requirements for enzyme preparations in the Food Chemicals Codex, 3d
ed. (1981), p. 110, 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, or
may be examined at the Office of Premarket Approval (HFS-200), Food and
Drug Administration, 200 C St. SW., Washington, DC, and the Office of
the Federal Register, 800 North Capitol St. NW., suite 700, 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.
3. Section 184.1034 is added to subpart B to read as follows:

Sec. 184.1034 Catalase (bovine liver).

(a) Catalase (bovine liver) (CAS Reg. No. 9001-05-2) is an enzyme
preparation obtained from extracts of bovine liver. It is a partially
purified liquid or powder. Its characterizing enzyme activity is
catalase (EC 1.11.1.6).
(b) The ingredient meets the general requirements and additional
requirements for enzyme preparations in the Food Chemicals Codex, 3d
ed. (1981), p. 110, 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 Office of Premarket Approval (HFS-
200), Food and Drug Administration, 200 C St., SW., Washington, DC, and
the Office of the Federal Register, 800 North Capitol St. NW., suite
700, 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 decompose hydrogen peroxide.
(2) The ingredient is used in food at levels not to exceed current
good manufacturing practice.
4. Section 184.1316 is added to subpart B to read as follows:

Sec. 184.1316 Ficin.

(a) Ficin (CAS Reg. No. 9001-33-6) is an enzyme preparation
obtained from the latex of species of the genus Ficus, which include a
variety of tropical fig trees. It is a white to off-white powder. Its
characterizing enzyme activity is that of a peptide hydrolase (EC
3.4.22.3).
(b) The ingredient meets the general requirements and additional
requirements for enzyme preparations in the Food Chemicals Codex, 3d
ed. (1981), p. 110, 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 Office of Premarket Approval (HFS-
200), Food and Drug Administration, 200 C St., SW., Washington, DC, and
the Office of the Federal Register, 800 North Capitol St., NW., suite
700, 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: [[Page 32911]]
(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.
5. Section 184.1415 is added to subpart B to read as follows:

Sec. 184.1415 Animal lipase.

(a) Animal lipase (CAS Reg. No. 9001-62-1) is an enzyme preparation
obtained from edible forestomach tissue of calves, kids, or lambs, or
from animal pancreatic tissue. The enzyme preparation may be produced
as a tissue preparation or as an aqueous extract. Its characterizing
enzyme activity is that of a triacylglycerol hydrolase (EC 3.1.1.3).
(b) The ingredient meets the general requirements and additional
requirements for enzyme preparations in the Food Chemicals Codex, 3d
ed. (1981), p. 110, 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 Office of Premarket Approval (HFS-
200), Food and Drug Administration, 200 C St., SW., Washington, DC, and
the Office of the Federal Register, 800 North Capitol St., NW., suite
700, 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 fatty acid glycerides.
(2) The ingredient is used in food at levels not to exceed current
good manufacturing practice.
6. Section 184.1443a is added to subpart B to read as follows:

Sec. 184.1443a Malt.

(a) Malt is an enzyme preparation obtained from barley which has
been softened by a series of steeping operations and germinated under
controlled conditions. It is a brown, sweet, and viscous liquid or a
white to tan powder. Its characterizing enzyme activities are
-amylase (EC 3.2.1.1.) and -amylase (EC 3.2.1.2).
(b) The ingredient meets the general requirements and additional
requirements for enzyme preparations in the Food Chemicals Codex, 3d
ed. (1981), p. 110, 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 Office of Premarket Approval (HFS-
200), Food and Drug Administration, 200 C St., SW., Washington, DC, and
the Office of the Federal Register, 800 North Capitol St., NW., suite
700, 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 starch or starch-derived
polysaccharides.
(2) The ingredient is used in food at levels not to exceed current
good manufacturing practice.
7. Section 184.1583 is added to subpart B to read as follows:

Sec. 184.1583 Pancreatin.

(a) Pancreatin (CAS Reg. No. 8049-47-6) is an enzyme preparation
obtained from porcine or bovine pancreatic tissue. It is a white to tan
powder. Its characterizing enzyme activity that of a peptide hydrolase
(EC 3.4.21.36).
(b) The ingredient meets the general requirements and additional
requirements in the Food Chemicals Codex, 3d ed. (1981), p. 110, 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
Office of Premarket Approval (HFS-200), Food and Drug Administration,
200 C St. SW., Washington, DC, and the Office of the Federal Register,
800 North Capitol St. NW., suite 700, 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.
8. Section 184.1595 is added to subpart B to read as follows:

Sec. 184.1595 Pepsin.

(a) Pepsin (CAS Reg. No. 9001-75-6) is an enzyme preparation
obtained from the glandular layer of hog stomach. It is a white to
light tan powder, amber paste, or clear amber to brown liquid. Its
characterizing enzyme activity is that of a peptide hydrolase (EC
3.4.23.1).
(b) The ingredient meets the general requirements and additional
requirements for enzyme preparations in the Food Chemicals Codex, 3d
ed. (1981), p. 110, 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 Office of Premarket Approval (HFS-
200), Food and Drug Administration, 200 C St. SW., Washington, DC, and
the Office of the Federal Register, 800 North Capitol St. NW., suite
700, 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.
9. Section 184.1914 is added to subpart B to read as follows:

Sec. 184.1914 Trypsin.

(a) Trypsin (CAS Reg. No. 9002-07-7) is an enzyme preparation
obtained from purified extracts of porcine or bovine pancreas. It is a
white to tan amorphous powder. Its characterizing enzyme activity is
that of a peptide hydrolase (EC 3.4.21.4).
(b) The ingredient meets the general requirements and additional
requirements for enzyme preparations in the Food Chemicals Codex, 3d
ed. (1981), p. 110, 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 Office of Premarket Approval (HFS-
200), Food and Drug Administration, 200 C St. SW., Washington, DC, and
the Office of the Federal Register, 800 North Capitol St. NW., suite
700, Washington, DC.
(c) In accordance with Sec. 184.1(b)(1), the ingredient is used in
food with no limitation other than current good
[[Page 32912]] 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: June 14, 1995.
Fred. R, Shank,
Director, Center for Food Safety and Applied Nutrition.
[FR Doc. 95-15239 Filed 6-23-95; 8:45 am]
BILLING CODE 4160-01-P}}}}}