Direct Food Substances Affirmed as Generally Recognized as Safe: Cellulase Enzyme Preparation Derived From Trichoderma Longibrachiatum for Use In Processing Food

DEPARTMENT OF HEALTH AND HUMAN SERVICES

Food and Drug Administration

21 CFR Part 184

[Docket No. 79G-0372]

Direct Food Substances Affirmed as Generally Recognized as Safe:

Cellulase Enzyme Preparation Derived From Trichoderma Longibrachiatum

for Use In Processing Food

AGENCY: Food and Drug Administration, HHS.

ACTION: Final rule.

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SUMMARY: The Food and Drug Administration (FDA) is amending its

regulations to affirm that cellulase enzyme preparation derived from

Trichoderma longibrachiatum (formerly called Trichoderma reesei) as

generally recognized as safe (GRAS) is for use in processing food. This

action is in response to a petition filed by the AAC Consulting Group,

Inc., on behalf of Novo Laboratories, Inc.

DATES: This regulation is effective May 26, 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 a certain

publication in Sec. 184.1250 (21 CFR 184.1250), effective May 26, 1999.

FOR FURTHER INFORMATION CONTACT: Nega Beru, Center for Food Safety and

Applied Nutrition (HFS-206), Food and Drug Administration, 200 C St.

SW., Washington, DC 20204, 202-418-3097.

SUPPLEMENTARY INFORMATION:

I. Background

In accordance with the procedures described in Sec. 170.35 (21 CFR

170.35), AAC Consulting Group, Inc. (formerly Arthur A. Checci, Inc.),

7445 Wisconsin Ave., suite 850, Bethesda MD 20814, on behalf of Novo

Nordisk BioChem North America, Inc. (formerly Novo Laboratories, Inc.),

State Rd. 1003, P.O. Box 576, Franklinton, NC 27525-0576, submitted a

petition (GRASP 9G0260) requesting affirmation that cellulase enzyme

preparation derived from a nonpathogenic strain of T. reesei (later

renamed T. longibrachiatum) used for processing food is GRAS.

Cellulase, the enzyme, is to be distinguished from cellulase enzyme

preparation, which contains cellulase as the principal active

component, but it also contains other components derived from the

production organism and fermentation media. This document will refer to

the former as ``cellulase'' and the latter as ``cellulase enzyme

preparation.''

In the Federal Register of November 27, 1979 (44 FR 67731), FDA

published a notice of filing of GRASP 9G0260, and gave interested

parties an opportunity to submit comments. FDA received one comment in

response to the notice. The comment urged the agency to affirm the GRAS

status of the cellulase enzyme preparation without restricting its use

in food other than to require that the use of the enzyme be consistent

with current good manufacturing practice.

II. Standards for GRAS Affirmation

Under Sec. 170.30 (21 CFR 170.30), 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 added to

food. The basis of such views may be either : (1) Scientific

procedures, or (2) in the case of a substance used in food prior to

January 1, 1958, experience based on common use in food

(Sec. 170.30(a)). 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

regulation 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.

In its petition, Novo Nordisk BioChem North America, Inc., relied

on scientific procedures, primarily published studies, scientific

papers and books, to demonstrate the safety and identity of the

cellulase enzyme and the production strain from which it is derived.

The petitioner provided published studies documenting that cellulase

enzyme preparation derived from nontoxicogenic, nonpathogenic T.

longibrachiatum is GRAS.

In evaluating this petition, the agency reviewed information

concerning: (1) The production organism, (2) the identity and function

of the cellulase enzyme, (3) the production and purification of the

cellulase enzyme preparation, (4) the use of the cellulase enzyme

preparation in the production of food products, and (5) the safety of

the enzyme preparation.

III. Safety Evaluation

A. Introduction

Commercial enzyme preparations that are used in food processing

typically are not chemically pure, but they contain, in addition to the

enzyme component, other components that derive from the production

organism and fermentation media, residual amounts of processing aids,

and substances used as stabilizers, preservatives or diluents. Issues

relevant to a safety evaluation of the enzyme preparation therefore

include the safety of the enzyme component, the safety of the enzyme

source, and the safety of processing aids and other substances added

during the manufacturing process. A safety evaluation of an enzyme

preparation also includes consideration of dietary exposure to that

preparation.

B. Production Organism

In a submission dated December 7, 1988, the petitioner informed

the agency that the International Commission on Taxonomy of Fungi

(ICTF) had decided to rename the source organism, a fungus known for

its high cellulase productivity, from T. reesei, to T. longibrachiatum

(Ref. 1). The petitioner presented published studies to assess

potential pathogenicity of T. longibrachiatum in mice, rabbits, and

guinea pigs (Ref. 2). No adverse reactions were reported in these

studies. The petitioner also included in its petition the results of a

search of several scientific data bases including Biological Abstracts,

1977-83; Chemical Abstracts, 1977-83; Scisearch, 1978-83; Medline,

1980-83; and Food Science and Technology Abstracts, 1969-83. The

petitioner states that these searches demonstrate that T.

longibrachiatum is well known and available to the scientific

community, and the data bases contain studies in which the

microorganism, or enzymes derived from it, were utilized without any

evidence of pathogenicity or toxicogenicity being associated with their

use. The searches did not identify a single report that T.

longibrachiatum is the etiological agent of a disease in man or

animals. The agency concludes, based upon the published information

presented in the petition (Refs. 2 through 6) that the production

organism T. longibrachiatum has been adequately identified and

determined to be nontoxicogenic and nonpathogenic (Ref. 7).

C. Identity and Function of the Cellulase Enzyme

Cellulase is the accepted name for the enzyme that catalyzes the

endohydrolysis of 1,4-beta-glucosidic linkages in cellulose (Ref. 8).

The enzyme will also hydrolyze 1,4-linkages in beta-glucans. The

enzymatically formed reaction products are mainly glucose and

cellobiose, a disaccharide composed of two glucose molecules. According

to the recommendations of the International Union of Pure and Applied

Chemistry and the International Union of Biochemistry (1972), cellulase

has the following designation: Cellulase, E.C. 3.2.1.4 (Ref. 9). FDA

concludes that generally available and accepted data and information

establish that the cellulase that is the subject of this document is

capable of achieving its intended technical effect.

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D. Production of Cellulase Enzyme Preparation

The production process for cellulase enzyme preparation from T.

longibrachiatum is described in GRASP 9G0260 and can be summarized as

follows. A pure culture of T. longibrachiatum is aseptically grown in a

typical culture medium such as one containing potato starch, soybean

meal, corn steep liquor, or dextrose. Mineral salts, such as phosphates

and sulfates, are included in the medium which also contains an

antifoaming agent and a surfactant. The fermentation is conducted at 26

to 32 deg.C with aeration and maximal agitation. Cell growth and the

possible presence of foreign microorganisms are monitored by taking

samples before inoculation of the fermenter, every 24 hours during

cultivation, and before transfer/harvesting.

After 100 to 170 hours, the culture broth is subjected to

flocculation and filtration. The enzyme, which is secreted into the

extracellular medium, is separated from the mycelium by action of the

flocculating agent. This material is then removed by filtration using a

filter aid. The enzyme, which remains in solution, is concentrated by

ultrafiltration or vacuum evaporation at 30 to 40 deg.C. The enzyme

suspension is then dried to a powder by spray drying or concentrated in

liquid form by vacuum evaporation. The packaged finished product,

powder or liquid, is shipped or stored at 4 deg.C.

The agency finds that the fermentation generating organism is

maintained in a manner to avoid contamination and genetic changes, that

the fermentation is a pure culture fermentation initially and is

monitored for purity periodically during the culture period, and that

the filtration step in the purification process would remove any viable

production organisms from the final product (Ref. 7). The agency

further finds that, because the potential impurities in the cellulase

enzyme preparation that may originate from the source or manufacturing

process do not raise any basis for concern about the safe use of the

preparation, the general requirements for enzyme preparations as

described in the ``Food Chemicals Codex,'' 4th ed. (1996) (Ref. 10),

which are being incorporated by reference in new Sec. 184.1250 in

accordance with 5 U.S.C. 552(a) and 1 CFR part 51, are adequate as

minimum criteria for food-grade cellulase enzyme preparation.

E. Use in Food

The function of cellulase enzyme preparation in food production

includes uses such as the breakdown of the cellulose in citrus

products, removal of fiber from edible oil press cakes, increase in

starch recovery from potatoes, extraction of proteins from leaves and

grasses, tenderizing fruits and vegetables prior to cooking, extraction

of essential oils and flavoring material from plant materials, the

preparation of animal feeds, and other uses that are discussed in

publications such as the Handbook of Food Additives (Refs. 11 and 12) .

The petitioner also presented additional published information

that the cellulase enzyme preparation performed its intended technical

effect in the production of various food materials. Cellulase enzyme

preparation has been shown to be effective in the degradation of

vegetable tissues and in the extraction of green tea components,

vegetable proteins and starches. Cellulase enzyme preparation is also

capable of modifying food materials such as vegetables, rice, and

soybeans to markedly influence the digestibility, cooking quality,

shape, and the yield of nutrients (Ref. 13).

The agency has considered the estimated dietary exposure to

cellulase enzyme preparation from its proposed use (Refs. 14 and 15).

Enzymes, including the petitioned cellulase, are used in small

quantities in food to accomplish their intended effects. In addition,

many food processes that use cellulase also include removal of

insoluble solids, a processing step that should remove most of the

added enzyme preparation. Nonetheless, in calculating the estimated

dietary exposure to cellulase enzyme preparation, the agency made the

conservative assumptions that no cellulase enzyme preparation is

removed from the food by processing, and all foods that may be treated

with cellulase enzyme preparation will be so treated. The agency

concludes that the dietary exposure to cellulase enzyme preparation

does not present a basis for concern about the safety of its use (Refs.

16 and 17).

F. Safety Studies

The petitioner has provided published studies with the cellulase

enzyme preparation, corroborated with unpublished studies, to

demonstrate that the enzyme preparation is safe for use in food. The

petitioner provided published oral acute toxicity studies with mice,

rats, and dogs and oral subchronic studies with rats and dogs (Ref. 2).

No significant adverse effects were noted in these studies.

A published toxicity study with in utero exposure, and a

teratogenicity study, both conducted with rats, reported no adverse

effects at levels up to 5 percent in the diet (Ref. 2). The petitioner

also provided published mutagenicity studies involving the Ames test,

chromosomal aberration tests, and dominant lethal tests (Ref. 2). There

was no evidence of mutagenicity of the cellulase enzyme preparation in

any of these tests. Other published studies with the cellulase enzyme

preparation provided by the petitioner include an inhalation study in

rats; skin and eye irritation tests in rabbits; a skin irritation test

in humans; and a skin sensitivity test in guinea pigs and humans.

Finally, because certain species of Trichoderma are known to produce

substances that inhibit the growth of microorganisms, the petitioner

tested the culture broth of T. longibrachiatum for antibiotics or

toxins; the tests were negative (Ref. 2).

The agency has reviewed the published safety studies in the

petition along with other available information. The agency concludes

that the published safety data support the use of cellulase enzyme

preparation from T. longibrachiatum for the enzymatic breakdown of

cellulose in processing food (Refs. 16 and 17).

IV. Conclusions

The agency has evaluated all available information and finds,

based upon the published information about the identity and function of

cellulase, that the enzyme component of cellulase enzyme preparation

will achieve its intended technical effect and raises no toxicity

concerns. The agency further finds, based upon generally available and

accepted information, that when the cellulase enzyme preparation is

manufactured in accordance with Sec. 184.1250, the source, T.

longibrachiatum, and the manufacturing process will not introduce

impurities into the preparation that may render its use unsafe. Finally

the agency finds that dietary exposure to the cellulase enzyme

preparation from the petitioned use does not present a basis for

concern about the safe use of the cellulase enzyme preparation.

Therefore, the agency concludes, based on the evaluation of published

data and information, and based upon scientific procedures

(Sec. 170.30(b)), that use of the cellulase enzyme preparation derived

from T. longibrachiatum for the enzymatic breakdown of cellulose in

processing food is GRAS. Therefore, the agency is affirming that the

use of

[[Page 28361]]

cellulase enzyme preparation from T. longibrachiatum described in the

regulation set out below is GRAS (21 CFR 184.1(b)(1)) with no

limitations other that current good manufacturing practice.

V. Environmental Effects

The agency has carefully considered the potential environmental

effects of this action. FDA has concluded that the action will not have

a significant impact on the human environment, and that an

environmental impact statement is not required. The agency's findings

of no significant impact and the evidence supporting these findings,

contained in an environmental assessment, may be seen in the Dockets

Management Branch (HFA-305), Food and Drug Administration, rm. 1061,

Rockville, MD 20852, between 9 a.m. and 4 p.m., Monday through Friday.

VI. Analysis of Impacts

A. Analysis for Executive Order 12866

FDA has examined the impacts of this final rule under Executive

Order 12866. Executive Order 12866 directs 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 if it

raises 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, it has been 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 substance. 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.

B. 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 Federal agencies to consider alternatives that would

minimize the economic impact of their regulations on small entities. In

compliance with the Regulatory Flexibility Act, FDA finds that this

final rule will not have a significant impact on a substantial number

of 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.

VII. 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.

VIII. 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)).

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. Cannon, P. F., ``International Commission on the Taxonomy of

Fungi (ICTF): Name Changes in Fungi of Microbiological Industrial

and Medical Importance. Part 2,'' Microbiological Sciences, 3(9):

285 to 287, 1986.

2. Hjortkjaer, R. K. et al., ``Safety Evaluation of Celluclast

TM, an Acid Cellulase Derived From Trichoderma reesei,'' Food

Chemical Toxicity, 21 (1) 55 to 63, 1986.

3. ``Specifications for the Identity and Purity of Some Enzymes

and Certain Other Substances'' in the Fifteenth Report of the Joint

FAO/WHO Expert Committee on Food Additives; WHO Food Additive

Series, No. 2, pp. 3 to 5, 1972 .

4. Rifai, M. A., ``A Revision of the Genus Trichoderma'' in

Mycological Papers, No. 116, pp. 42 to 44, 1969.

5. Simmons, E. G., ``Classification of Some Cellulase Producing

Trichoderma Species'' in Second International Mycological Congress,

Book of Abstracts, p. 618, 1977.

6. Church, B. D., N. A. Nash, and W. Brosz, ``Use of Fungi

Imperfecti in Treating Food Processing Wastes,'' in Development in

Industrial Microbiology, vol. 13, pp. 30 to 46, 1972.

7. Memorandum from Food and Cosmetics Microbiology Branch, FDA,

to GRAS review Branch, FDA, January 10, 1980.

8. King, K. W., and M. I. Vassal, ``Enzymes of the Cellulase

Complex,'' in Cellulase and Their Applications, edited by R. F.

Gould ``Advances in Chemistry Series'' #95, American Chemical

Society, Washington, DC, pp. 7 to 25, 1969.

9.``Enzyme Nomenclature,'' recommendations (1972) of the

International Union of Pure and Applied Chemistry and the

International Union of Biochemistry, American Elsevier, New York,

pp. 212 to 213, 1975.

10. ``Monograph on Enzyme Preparations'' in Food Chemicals

Codex, 4th ed., National Academy Press, Washington, DC, pp. 129 to

134, 1996.

11. Underkofler, L. A., ``Enzymes'' in CRC Handbook of Food

Additives, edited by T. E. Furia, The Chemical Rubber Co., Ohio, pp.

80 to 82, 1968.

12. Malmos, H., ``Industrial Applications of Cellulase: Enzyme

Applications in Food, Pharmaceuticals and Other Industries,'' in

American Institute of Chemical Engineers Symposium Series, vol. 74,

1976/77, pp. 93-99.

13. Toyoma, N., ``Applications of Cellulases in Japan,'' pp.

359 to 390, in Cellulases and Their Applications, edited by R. F.

Gould, Advances in Chemistry Series 195, American Chemical Society,

Washington, DC, 1969.

14. Memorandum from Food and Color Additives Review Section,

FDA, to Direct Additives Branch, FDA, February 21, 1989.

15. Memorandum from Food and Color Additives Review Section,

FDA, to Direct Additives Branch, FDA, June 22, 1990.

16. Memorandum from Additives Evaluation Branch, FDA, to Direct

Additives Branch, FDA, July 11, 1990.

17. Memorandum from Additives Evaluation Branch, FDA, to Direct

Additives Branch, FDA, June 29, 1993.

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 SUBSTANCE 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 184.1250 is added to subpart B to read as follows:

Sec. 184.1250 Cellulase enzyme preparation derived from Trichoderma

longibrachiatum.

(a) Cellulase enzyme preparation is derived from a nonpathogenic,

nontoxicogenic strain of Trichoderma

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longibrachiatum (formerly T. reesei). The enzyme, cellulase, catalyzes

the endohydrolysis of 1,4-beta-glycosidic linkages in cellulose. It is

obtained from the culture filtrate resulting from a pure culture

fermentation process.

(b) The ingredient meets the general and additional requirements

for enzyme preparations in the monograph specifications on enzyme

preparations in the ``Food Chemicals Codex,'' 4th ed. (1996), pp. 129

to 134, 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., Box 285, Washington, DC

20055 (Internet ``http://www.nap.edu''), 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 St. NW., suite 700, Washington, DC.

(c) In accordance with Sec. 184.1(b)(1), the ingredient is used in

food with no limitation other than current good manufacturing practice.

The affirmation of this ingredient as generally recognized as safe

(GRAS) as a direct human food ingredient is based upon the following

current good manufacturing practice conditions of use:

(1) The ingredient is used in food as an enzyme as defined in

Sec. 170.3(o)(9) of this chapter for the breakdown of cellulose.

(2) The ingredient is used in food at levels not to exceed current

good manufacturing practice.

Dated: May 17, 1999.

L. Robert Lake,

Director, Office of Policy, Planning and Strategic Initiatives, Center

for Food Safety and Applied Nutrition.

[FR Doc. 99-13151 Filed 5-25-99; 8:45 am]

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