Enzyme Preparations From Animal and Plant Sources; Affirmation of Gras Status as Direct Food Ingredients

Federal RegisterJun 26, 1995

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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

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

activities3 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 similar4 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 preparation5. 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.

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.

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.

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).