[Federal Register Volume 64, Number 78 (Friday, April 23, 1999)]
[Rules and Regulations]
[Pages 19887-19895]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 99-10011]


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DEPARTMENT OF HEALTH AND HUMAN SERVICES

Food and Drug Administration

21 CFR Part 184

[Docket No. 84G-0257]


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

AGENCY: Food and Drug Administration, HHS.

ACTION: Final rule.

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

 DATES: The regulation is effective April 23, 1999. The Director of the 
Office of the Federal Register approves the incorporation by reference 
in accordance with 5 U.S.C. 552(a) and 1 CFR part 51 of certain 
publications listed in 21 CFR 184.1148 and 184.1150, effective April 
23, 1999.
FOR FURTHER INFORMATION CONTACT: Linda S. Kahl, Center for Food Safety 
and Applied Nutrition (HFS-206), Food and Drug Administration, 200 C 
St. SW., Washington, DC 20204, 202-418-3101.

SUPPLEMENTARY INFORMATION:

Table of Contents

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

I. Introduction

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

[[Page 19888]]

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

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

III. Background

A. Identity and Technical Effect

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

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

B. Methods of Manufacture

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

[[Page 19889]]

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

IV. Safety Evaluation

A. Pre-1958 History of Use in Food

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

  Table 2.--Applications of Bacterial Carbohydrase and Protease Enzyme
                   Preparations in Food Prior to 1958
------------------------------------------------------------------------
                                          Technical
      Enzyme                              effect or
   preparation       Food categories      industry          References
                                         application
------------------------------------------------------------------------
 Carbohydrase       Beer               Mashing1          4 and 5
                    Syrup for cocoa    Reduction of      4 and 5
                    and chocolate      viscosity         4 and 5
                    Sugar              Recovery from     4 and 5
                    Distilled          scrap candy       4
                    beverages          Mashing
                    Precooked          Modification of
                    cereals            cereal starches
                                       to improve
                                       characteristics
 Protease           Beer               Chillproofing     4
                    Condiments         Not reported      5
                    Milk               Protein           5
                                       hydrolysis
------------------------------------------------------------------------
\1\ Mashing is the conversion of starch to sugars.

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

[[Page 19890]]

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

B. Corroborating Evidence of Safety

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

[[Page 19891]]

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

[[Page 19892]]

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

V. Comments

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

[[Page 19893]]

VI. Conclusions

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

VII. Environmental Considerations

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

VIII. Analysis for Executive Order 12866

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

IX. Regulatory Flexibility Analysis

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

X. Paperwork Reduction Act of 1995

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

XI. Effective Date

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

XII. References

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

[[Page 19894]]

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

List of Subjects in 21 CFR Part 184

    Food additives, Food ingredients, Incorporation by reference.
    Therefore, under the Federal Food, Drug, and Cosmetic Act and under 
authority delegated to the Commissioner of Food and Drugs, and 
redelegated to the Director, Center for Food Safety and Applied 
Nutrition, 21 CFR part 184 is amended as follows:

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

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

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

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

Sec. 184.1148   Bacterially derived carbohydrase enzyme preparation.

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

[[Page 19895]]

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

Sec. 184.1150 Bacterially-derived protease enzyme preparation.

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

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