The Enzyme Technical Association (ETA) has created the guidelines contained in this booklet to assist the dietary supplement industry in safely producing high quality enzyme containing dietary supplement products. This document recommends guidelines for enzyme safety, toxicity, activity, shelf stability and other specifications to complement the United States Food and Drug Administration’s (FDA) current Good Manufacturing Practices (cGMPs) and other applicable regulations in regard to the production and safe handling of enzymes that are used in dietary supplements.

Enzymes have a long and safe history of use in human foods including their use as dietary supplements where they offer many benefits to the consumer. However, the ETA also recognizes that (1) the growth of the use of dietary supplements may result in wider consumer use and exposure to enzymes in forms and concentrations not previously experienced and (2) manufacturers with limited experience with bulk enzyme concentrates may become involved in the production of dietary supplements containing enzymes. The ETA desires to provide guidance for the production, handling or consumption of enzyme-containing dietary supplements.

In general, enzymes are unremarkable in terms of their toxicological profiles, i.e. they possess a low order of toxicity. Some types of enzymes, e.g. protease, are irritating to skin and eyes, particularly at high concentrations or upon prolonged contact. The main safety concern associated with enzymes is the induction of respiratory allergies among workers in the manufacturing environment where repeated exposure to foreign protein may induce sensitivity. Enzymes are proteins and like many other proteins foreign to the human body, they can stimulate the body’s immune system if inhaled at sufficient concentrations. The safety of enzymes commonly used in foods and food processing is well established.

The FDA is in the process of promulgating GMP regulations for dietary supplements (Advance Notice of Proposed Rulemaking, docket no. 96N-0417, RIN 0910-AA59, Current Good Manufacturing Practice in Manufacturing, Packing, or Holding Dietary Supplements). The enclosed guidelines are meant to complement the FDA GMPs and other regulations in areas unique and specific to enzymes. These guidelines are meant to complement cGMPs or other forms of guidance provided by FDA.

What are Enzymes

Enzymes are large proteins and, like other proteins, they are produced in living cells of plants, animals and micro-organisms. All living organisms require enzymes for growth and for the production and utilization of energy that is essential for life.

II. Risk Assessment

The potential for both consumer and workplace exposure associated with enzyme-containing dietary supplements dictates that the processes of risk assessment and risk management be a specific part of the development of new consumer products.

Risk assessment is the process of identifying the toxic/hazardous profile of a given material as it relates to human exposure, in order to gauge both the nature of potential adverse effects and the likelihood of their occurrence. This process is based on a series of questions that risk assessors ask about scientific information (hazard identification, dose-response assessment, human exposure assessment) that is relevant to human risk (ref. Fund. Appl. Tox., Vol 39, p.81, 1997). A carefully designed, properly executed risk assessment is important for enzyme-containing consumer products because it will help assure the safety of the product. Because of the importance of risk assessment and risk management as it relates to enzyme-containing consumer products, the ETA is co-operating with the Soap and Detergent Association (SDA) in the publication of a booklet that specifically addresses these issues and enzymes in detail. When this booklet is completed it can be obtained from SDA (New York, NY, 212-725-1262). Manufacturers of dietary supplements containing enzymes are strongly urged to obtain a copy of this document when it becomes available.

Each producer and marketer of enzyme-containing dietary supplements should conduct and keep on file the risk assessments they have completed.

III. Potential for Interactions and Adverse Effects

Enzymes may have the potential for interaction with other ingredients contained in the same supplement and also with other dietary supplements, drugs or medications that a consumer may be using. Such interactions could adversely affect the function of the enzyme itself or the function of other medications. Each producer and marketer of enzyme-containing dietary supplements should consider the potential for such interactions and/or adverse effects and make label advisements as appropriate.

IV. Safe Handling of Enzymes

Working safely with enzymes in the dietary supplements industry has much in common with enzyme safety practices in other industries. Reference to previously published information on this topic such as Working Safely With Enzymes (Enzyme Technical Association, 1995) and Work Practices for Handling Enzymes in the Detergent Industry (Soap and Detergent Association, 1995) is useful.

In recent years, the enzyme industry and the users of enzymes such as the detergent and food processing industries have moved away from the use of enzymes in dry, powdered forms. Instead forms such as liquids or granulates have been developed in order to limit the potential worker exposure to enzyme containing dust or aerosols. In the dietary supplements industry the use of dry forms other than granulates may be necessary for use in tableting or production of capsules. Therefore, special attention may be required to prevent exposure of workers to enzyme aerosols in the form of dust. This issue as well as general safe practices is addressed below.

Routes of Exposure and Possible Health Effects

When handling concentrated enzyme preparations, as with most substances used in industrial processes, care should be taken to avoid skin contact and inhalation of aerosols. Enzymes can be used safely without any adverse health effects through proper employee education, good work practices, engineering controls and appropriate personal protective equipment.

Irritation

Prolonged skin contact with proteolytic enzymes can cause skin irritation. The eyes can also be irritated by contact with proteolytic enzymes. As would be expected, the more concentrated the enzyme preparation, the greater the potential for producing irritation upon contact. Skin irritation is most likely to appear in body areas where perspiration occurs, i.e., hands, armpits, groin, and feet and around tight fitting clothing areas, such as cuffs, waist, collar, and facial areas in contact with face masks. This irritation is caused by the chemical properties of the proteases.

Other types of enzymes, i.e., non-proteolytic enzymes, have not been shown to cause skin and eye irritation. However, skin and eye contact with all enzymes should be minimized as part of good personal hygiene practices. As with any chemical, avoid contact with enzymes if the skin is broken or irritated. Consult the manufacturer’s Materials Safety Data Sheet (MSDS) for information on the hazards associated with other ingredients of the enzyme preparation.

Inhaling high levels of enzyme-containing aerosols may result in coughing and/or congestion due to irritation of the mucous membranes of the respiratory tract. Respiratory irritation is a very rare occurrence and can be prevented when adequate manufacturing controls are in place.

Allergy

As with any protein that is foreign to the respiratory tract, repeated inhalation of enzyme contained in aerosols can cause an allergic response. Predicting who will develop an allergic response or the level and duration of exposure needed to elicit a response is not known at this time.

As with any protein allergen, such as pollen, mild to severe symptoms may occur and may include any, or a combination of, the following: Asthma, sneezing, nasal or sinus congestion, coughing, watery eyes, runny nose, tightness of the chest, hoarseness or shortness of breath. These symptoms may develop during work hours or can be delayed, occurring even two or more hours after work exposure. Symptoms will occur only in an allergic individual if enzyme aerosols are inhaled, and usually disappear within hours or a few days after exposure is eliminated. Currently, there is no evidence to indicate that skin contact with enzymes will cause allergic contact dermatitis. Aside from allergies, no long-lasting effects from working with enzymes have been found.

Ordinary cold or flu symptoms may resemble enzyme allergy. If symptoms appear more often during working days, especially at the beginning of the work week and seldom or never on the weekends or holidays, they may be due to enzyme exposure, and this possibility should be investigated. A person exhibiting allergic symptoms should consult a physician.

Safe Handling Practices

When working with enzyme-containing preparations, it is important to use work practices that do not generate aerosols or that result in direct skin contact. For each work operation, careful consideration must be given to minimizing aerosol formation and skin or eye contact. Aerosols are defined as airborne solid or liquid substances such as dusts and mists.

Aerosols are formed through high-energy operations such as mixing, grinding, milling, washing with high water pressure or steam, and using compressed air for cleanup operations. Sweeping, blowing, splashing, steam cleaning, and high-pressure water flushing must be avoided. Mixing and grinding operations should be contained as much as possible, and the areas in which they take place should be provided with adequate local exhaust ventilation.

Unique to the dietary supplements industry are the practices involved in tableting, capsule production, packaging into small vials or packets, etc. These operations may introduce additional risks of exposure to workers and may present special challenges for containment and worker protection. These operations should be evaluated for exposure potential and engineering controls such as isolation and local exhaust ventilation. Wherever exposure to enzyme dust or aerosols may occur, personal protective equipment should be employed. At tableting and filling sites the process should be enclosed and supplemented with local exhaust ventilation. The ventilation systems should be properly vented outside of the building.

When handling enzyme preparations or enzyme-contaminated equipment, measures should be taken to avoid direct skin contact. Wear appropriate gloves and clothing when there is a potential for contact with enzymes. Wash enzyme-contaminated surfaces thoroughly before handling. Maintenance and cleaning operations should be evaluated and particular care taken to prevent exposure.

Care should be taken in equipment design in order to minimize spillage, dust and aerosol formation as well as operations that might result in the grinding or breakage of tablets or capsules. Bottling or packaging operations should also be enclosed as much as possible and supplemented with appropriate personal protection and local exhaust ventilation.

USE OF PERSONAL PROTECTIVE EQUIPMENT

Respiratory Protection

In facilities designed to assure containment, respiratory protection is not normally necessary, However, there are some operations, such as spill cleanup, equipment cleaning and equipment repairing, that may generate aerosols. In these instances, respiratory protection may be necessary. The use of respiratory protection is often necessary when working with powdered enzymes.

Respiratory protection should always be used when indicated by your supervisor, safety professional or medical personnel.

The Occupational Safety and Health Administration (OSHA) respiratory protection standard must be followed in the selection, training and use of respirators. Use only National Institute of Occupational Safety and Health (NIOSH) approved respiratory protection.

Protective Clothing and Gloves

Protective clothing should be worn when there is a potential for skin or eye contact. This clothing may include gloves, aprons, safety glasses, and outer garments, such as coveralls or lab coats. Protective clothing is particularly important when working with proteolytic enzymes, which are known to cause skin irritation. Operations that may require the use of protective clothing include spill cleanup, equipment maintenance, and equipment cleaning or when working with powdered enzymes. Gloves should be worn when there is a potential for skin contact with any enzyme material. Cotton liners or cotton-lined gloves are recommended to absorb perspiration. Protective clothing should be removed prior to leaving the work area and should not be worn into other areas of the facility (i.e., lunchroom, offices) or to the home.

The OSHA personal protective equipment standard (1910.132-138) must be followed in selection, training and use of personal protective equipment. Consult the enzyme manufacturer and/or their MSDS for additional information on the selection of personal protective equipment.

Maintenance

Whenever maintenance is to be performed on equipment that has been in contact with enzymes, the equipment should be thoroughly cleaned before the work is begun. Use wet washing (flooding, wiping) or a vacuum system equipped with a high-efficiency particulate air filter (HEPA) to clean equipment or spills. High-pressure cleaning (steam, air, or water) must be avoided, since these operations cause aerosol formation. Personal protective equipment (gloves, respirators, eye protection) may be required during some maintenance operations.

Spill Clean Up

Spilled enzymes must be removed immediately by a central vacuum system, vacuums equipped with a HEPA filter, mopping, or washing. To prevent dust or aerosol formation during cleanup, do not sweep or use high water pressure, steam, or compressed air on spills. Use plenty of water in wet washing to flush all enzyme material away to prevent enzyme dust generation from dried residual material. Dependent upon the place and extent of the spill, respiratory protection and protective clothing may be required during cleanup.

Disposal of spilled material should be in compliance with federal, state and local regulations.

Personal Cleanliness

Personal cleanliness is essential to prevent irritation from proteolytic enzymes to skin and mucous membranes and to avoid unnecessary spreading of the contaminants to other areas. The irritation response on skin is increased in the presence of moisture and when the natural oils of the skin are removed.

The following procedures are recommended to prevent irritation:

• Hands should be washed with water and mild soap before leaving the work area and immediately after coming into contact with enzyme materials.

• Change work clothes daily and whenever they are soiled with enzyme material. Do not wear work clothing home.

• Avoid touching your face and eyes with enzyme contaminated clothing or gloves.

• Wear cotton-lined gloves to absorb perspiration.

Measuring Enzyme Levels in Air

There are air monitoring techniques available to measure the level of enzyme dust or mist in the air. The American Conference of Governmental Industrial Hygienists (ACGIH) has established a threshold limit value (TLV) for only one class of enzymes, subtilisins, of 60 ng/m3 as a ceiling limit. Both low-flow and high-flow air sampling methods are available for some enzymes. Contact the enzyme manufacturer for additional information.

FIRST AID TREATMENT

Skin Contact

Most enzyme materials are water soluble; therefore, the exposed skin should first be thoroughly flushed with water and then washed with a mild soap and water. If clothes are contaminated, remove them, shower and change into clean clothes. Immerse the contaminated clothes in water and wash them as usual.

Inhalation

Remove the individual from exposure and monitor for irritation or allergic symptoms. If symptoms occur, consult a physician. Symptoms may not occur until two or more hours after exposure.

Eye Contact

Rinse the eyes thoroughly with water for at least 15 minutes and then consult a physician.

Summary

Through the use of proper work practices and control measures, enzymes can be handled in the work place without any adverse health effects. All work with enzymes must be done with care and proper precautions. Avoid generation of aerosols and direct skin or eye contact when handling enzyme materials. Even though there may be no visible signs of dust or aerosols, appropriate safety measures must be followed at all times. By following these relatively simple work practices and control measures, enzymes can be handled safely.

V. New Enzymes Used in Dietary Supplements

The Dietary Supplement Health and Education Act of 1994 (PL 103-417, DSHEA) defines "new dietary ingredients" as those dietary ingredients not marketed in the United States before October 15, 1994 (ibid. Sec 8c). Any enzyme, which was grandfathered as a dietary supplement prior to October 15, 1994, any enzyme which is Generally Recognized As Safe (GRAS) or any enzyme, which is the subject of and meets the criteria of a food additive petition may not require additional toxicological testing. A list of enzymes known to ETA member companies as having dietary use in the US prior to October 15, 1994, is attached as Appendix A.

For "new enzymes" to be used for dietary supplements, the ETA recommends the following testing should be performed on the enzyme preparation (test material):

1. Subchronic toxicity study of 90 days in a rodent species. The No Observable Adverse Effect Level (NOAEL) should be sufficiently high to assure safety*.

2. Mutagenicity studies using gene mutation in Salmonella typhimurium (Ames, B.N., J Mc Cann and E. Yamasaki, Methods for Detecting Carcinogens and Mutagens with the Salmonella / Mammalian-Microsome Mutagenicity Test, Mutation Research 31:347-364, 1975, and Maron, D. and B. Ames, Revised Methods for the Salmonella Mutagenicity Test, Mutation Research 113:173-215, 1983).

3. The chromosome aberration test.

*The phrase "sufficiently high" refers to appropriate multiples of the estimated mean human consumption level. For enzymes used in dietary supplements, the NOAEL should be at least 100 times the estimated mean human consumption level. Where the product is itself a food or a major food component or otherwise consumed in large quantities, it may not be possible to test at this high level. In these cases, safety may be established by feeding the highest level compatible with the maintenance of adequate nutritional requirements in consideration of the questions outlined in the decision tree for whole foods and complex mixtures presented in Figure 7 of Regulatory Toxicology and Pharmacology 12:S136-S158(1990).

The prescribed toxicity tests noted above are to be conducted with enzyme preparations as the test material, not as a finished product for the consumer. Reference is also made to the discussion of risk assessment which appeared earlier in this booklet.

VI. Guidelines for Enzyme Specifications

In the United States, DSHEA has specifically defined dietary supplements as food (op. cit. sec. 3a). It is therefore recommended that enzymes used as dietary supplements meet the specifications for food enzymes selected from the Food Chemicals Codex (FCC), Fourth Edition (1996), pp. 133-134. (National Academy Press, 2101 Constitution Avenue, N.W., Washington, D.C. 20118.), the Joint FAO/WHO Expert Committee on Food Additives in FAO Food and Nutrition Paper No. 49, pp. 80-83, and the Reports of the Scientific Committee for Food: 27th series, pp. 13-32.

While these requirements are summarized below, the dietary supplement manufacturer should review the referenced documents to be certain that all aspects relevant to their product(s) are addressed:

GENERAL REQUIREMENTS

Enzyme preparations shall be produced in accordance with current good manufacturing practices. Regardless of the source from which they are derived, they should cause no increase in the total microbial count in the treated food over the level accepted for the respective food.

Animal tissues used for the production of enzymes must comply with the applicable U.S. meat inspection requirements and must be handled with good hygienic practices.

Plant material used to produce enzymes or culture media used to grow micro-organisms shall consist of components that leave no residue harmful to health in the finished food under normal conditions of use.

Preparations derived from microbial sources shall be obtained using a pure culture fermentation of a non-pathogenic and non-toxigenic strain and are produced by methods and under culture conditions that ensure a controlled fermentation, thus preventing the introduction of micro-organisms that could be the source of toxic materials and other undesirable substances.

The carriers, diluents, and processing aids used in the production of the enzyme preparations shall be substances that are acceptable for general use in foods, including water and substances that are insoluble in foods, but removed from the foods after processing.

Although tolerances have not been established for mycotoxins, appropriate measures should be taken to ensure that the products do not contain such contaminants.

ADDITIONAL REQUIREMENTS

Limit Method

Reference

Assay Not less than 85.0% and not more than FCC

115.0% of the declared activity.

Heavy Metals (as Pb) Not more than 30 mg/kg. FCC

Lead Not more than 5 mg/kg. FCC

Microbial Limits:

Coliforms Not more than 30 per g. FCC

E. coli Negative in 25 g. JECFA BAM

Salmonella sp. Negative in 25 g. FCC

Listeria Negative in 25 g. JECFA BAM

Total Viable Count Not more than 3000/g JECFA BAM

Yeasts & Mold Not more than 100/g JECFA BAM

Antibiotic Activity not detected JECFA JECFA

Mycotoxins and Aflatoxins not detected FCC AOAC

VII. ENZYME ACTIVITY METHODS FOR DIETARY SUPPLEMENTS

In order for consumers and health professionals to be able to compare enzymes, both in the commercial market place and in clinical and other published studies, it is necessary that enzyme potencies be expressed in scientifically sound units of activity.

The ETA guidelines for enzyme activities are:

1. Enzymes in dietary supplements should be measured and labeled on an activity basis, not a weight basis.

2. An authorized, compendial method of measuring and expressing enzyme activity such as Food Chemicals Codex (FCC), United States Pharmacopoeia (USP), Federation Internationale Pharmaceutique (FIP) or Japan Pharmacopoeia (JP) should be adopted whenever possible.

3. For enzymes not adequately covered in compendial sources, it is recommended that activity methods that are used have undergone scientifically sound development and validation procedures.

4. When developing new assay methods, widely available equipment and reagents should be used.

5. The assay temperature should be body temperature, 37oC, unless characteristics of the enzyme preclude this temperature.

6. An assay pH range of pH 4 to pH 5 is recommended whenever possible if the enzyme is to be ingested.

7. A well-defined substrate with adequate lot-to-lot uniformity should be used.

8. At a minimum, validation of enzyme assays should document assay specificity, assay variability, assay linearity and assay sensitivity.

VIII. SHELF STABILITY

Producers and marketers of dietary supplements containing enzymes must recognize that enzymes can be labile if exposed to unfavorable conditions during formulation, manufacture, storage, distribution, sale or home storage. Parameters such as product ingredients, initial moisture content, hygoscopicity of ingredients, ambient temperatures, ambient humidity, product packaging and time can all impact enzyme stability. It is the producer’s and marketer’s responsibility to assure that label claims for activities are met and that shelf life or expiration dates, if used, accurately reflect enzyme stabilities.

ETA Guidelines:

1. In the case of blended enzymes or enzymes blended with excipients which prevent adequate activity testing, the individual enzymes or the enzyme without the offending excipient shall be used to determine the final activity.

2. Product shall be formulated to contain not less than 100% of the declared or labeled activity.

3. Final product should be assayed to contain plus or minus 15% of the declared or labeled activity.

4. Appropriate accelerated stability studies or data from similar product formulations may be used for an initial determination of shelf life.

5. Recommended conditions for accelerated stability testing are 40oC and 75% relative humidity.

6. Product shelf life shall be confirmed and may be extended on the basis of real time studies on products stored under labeled or recommended storage conditions.

7. Final product shelf stability shall be confirmed with samples from at least three representative lots of product.

8. The enzymes used in stability tests must be in their final form, e.g. tablet, capsule, powder, etc., for consumer use.

9. The enzymes used in stability tests must be in the final, sealed container.

10. The declared shelf stability will be no longer than the point that a minimum of three different production lots of the enzyme, meeting the above criteria, retain 85% of the labeled or declared activity.

11. Shelf stability studies should continue on future production lots to ensure that shelf stability continues to meet specifications.

Conclusion

The information in this booklet is provided by ETA to assist the dietary supplement industry in the safe development and handling of products that contain enzymes. These guidelines are intended to complement the forthcoming FDA GMP regulations for dietary supplements. Should a conflict between FDA GMP regulations and ETA guidelines exist, we propose that the more stringent of the two apply.

It is hoped that the information provided in this booklet will help answer some of the questions about enzymes, how they may be used in dietary supplements and how to work with them safely. If you should have further questions, consult your enzyme supplier.

APPENDIX A

ENZYMES IN USE FOR DIETARY PURPOSES IN THE U.S.

PRIOR TO OCTOBER, 1994*

Enzyme Origin Enzyme Origin

Alpha-galactosidase Aspergillus niger

Amylase

Bacillus amyloliquefaciens

Aspergillus oryzae

Aspergillus niger

Amylase (Malt diastase) malt

Amyloglucosidase

Rhizopus oryzae

Beta-amylase

wheat

Bacillus spp.

Catalase Aspergillus niger

Cellulase

Aspergillus niger

Trichoderma longibrachiatum (reesei)

Glucose oxidase Aspergillus niger

Hemicellulase

Aspergillus niger

Trichoderma longibrachiatum (reesei)

Bacillus subtilis

Invertase Saccharomyces cerevisiae

Lactase

Aspergillus oryzae

Kluveromyces lactis

Lipase

Rhizomucor miehei

Rhizopus oryzae

Rhizopus delemar

Lysophospholipase Aspergillus niger

Lysozyme egg white

Pancreatin Porcine pancreas

Pancrelipase Bovine and porcine pancreas

Pectinase

Rhizopus oryzae

Phospholipase porcine

Phytase Aspergillus niger

Protease, microbial

Aspergillus oryzae

Aspergillus niger

Aspergillus melleus

Bacillus licheniformis

Bacillus thermoproteolyticus

Rhizopus niveus

Protease, botanical

papaya latex (papain)

pineapple, root and stem (bromelain)

Protease, animal

bovine or porcine (trypsin)

bovine or porcine (chymotrypsin)

bovine or porcine (pepsin)

rennin

SuperOxide Dismutase Bacillus spp.

* As provided by member companies of the Enzyme Technical Association.