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 supplements. This document recommends guidelines for enzyme safety, product safety evaluation, activity, shelf life and other parameters to complement, but not replace, 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. The ETA calls attention to the fact that on June 25, 2007 US - FDA published the dietary supplement GMPs in the Federal Register (21 CFR Part 111) http://edocket.access.gpo.gov/2007/07-3039.htm. While dates for compliance range from June 25, 2008 to June 25, 2010, depending upon the size of the manufacturer, ETA encourages anyone involved in the manufacture of Dietary Supplements to review the new GMP's.

Enzymes have a long history of safe use in human foods including their use in 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. It is the intention of the ETA to provide guidance for the production, handling and consumption of enzyme-containing dietary supplements.

In general, enzymes are unremarkable in terms of their toxicological profiles in that they possess a low order of toxicity. Proteases are irritating to skin and eyes, particularly at high concentrations or upon prolonged contact. The main safety concern associated with enzymes is the occurrence of respiratory allergies among workers in the manufacturing environment where repeated exposure to aerosolized enzyme protein may induce the development of allergenic antibodies. Enzymes are proteins and like many other proteins foreign to the human body can act as antigens and stimulate the body's immune system if inhaled. ETA encourages a sound worker safety program in the manufacturing environment to limit exposure to high concentrations of enzyme protein dusts and aerosols.

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

Enzymes have been used safely in the production of food for centuries. However, there is potential for consumer and workplace exposure when handling enzymes. ETA therefore, advises manufacturers of enzymes and dietary supplements that incorporate enzymes to follow the processes of risk assessment and risk management during the development of new consumer products.

Risk assessment is the process of identifying the hazard 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 co-operated with the Soap and Detergent Association (SDA) in the publication of a booklet, Risk Assessment Guidance of Enzyme-Containing Products. This publication specifically addresses these issues and enzymes in detail and can be obtained from SDA (+202-347-2900). ETA and SDA encourage manufacturers of dietary supplements containing enzymes to obtain a copy of this document.

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 affect the function of the enzyme itself or of other medications. Though enzymes are not usually considered allergenic by ingestion, an allergic response to the enzyme protein is possible in hypersensitive individuals. Each producer and marketer of enzyme-containing dietary supplements should be familiar with all ingredients and consider the potential for 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.

Unique to the dietary supplements industry are the practices involved in tabletting, capsule production and packaging into small vials or packets, etc. These operations may introduce the risk of exposure to workers and may present special challenges for containment and worker protection. These operations should be evaluated in advance for potential exposure and possible need for engineering controls such as isolation and local exhaust ventilation. Wherever exposure to enzyme dusts or aerosols may occur, personal protective equipment should be employed. At tabletting 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 taking into account all federal, state and local regulations.

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" considered for use in dietary supplements, the ETA recommends the following testing should be performed on the enzyme preparation (test material):

1. Subchronic toxicity study (90 days) in a rodent species. The No Observed Adverse Effect Level (NOAEL) should be sufficiently high to assure safety*.
2. Bacterial reverse mutation test (OECD, 1997 - OECD Guideline for the testing of Chemicals No. 471: Bacterial Reverse Mutation Test. Adopted 21 July 1997)
3. The chromosome aberration test (OECD, 1997 - OECD guideline for the testing of chemicals No. 473: In vitro mammalian chromosome aberration test. Adopted 21 July 1997)

*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 daily maximum human consumption level. Where the enzyme product is 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 recommended toxicity tests noted above are to be conducted using enzyme concentrates or preparations as the test material rather than using the finished product intended for the consumer. Reference is also made to the discussion of risk assessment which appeared earlier in this booklet. The safety evaluation concepts in Pariza and Foster (1983) and Pariza and Johnson (2001) should also be followed.

VI. Guidelines for Enzyme Specifications

Enzyme preparations should be produced in accordance with appropriate current good manufacturing practices. Guidelines governing the specifications required for production and process control under the dietary supplement GMPs are listed in 21 CFR Part III, Subpart E (111.70). They include establishing the following:

1. An identity specification
2. Specifications for components of the dietary supplement that are necessary to ensure the purity, strength and composition
3. Identification of and limits on the types of contamination that may adulterate or lead to adulteration of the finished batch of the dietary supplement to ensure the quality of the supplement

Regardless of the source from which they are derived, enzyme preparations should not contain any viable cells of the production organism.

GENERAL REQUIREMENTS

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 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. This includes (potable) water and substances that are insoluble in foods, but are removed from the foods after processing.

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

VII. ENZYME ACTIVITY METHODS FOR DIETARY SUPPLEMENTS

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 rather than a weight basis, if possible.
2. An authorized, compendial method of measuring and expressing enzyme activity such as would be found in the Food Chemicals Codex (FCC), United States Pharmacopoeia (USP), Federation Internationale Pharmaceutique (FIP) or Japan Pharmacopoeia (JP) should be adopted whenever possible.
3. For enzymatic activity not adequately covered in compendial sources, it is recommended that methods to measure activity have undergone scientifically sound development and validation procedures to ensure accuracy and reproducibility. When developing new methodology, the substrate pH should be representative of the target section of the gastrointestinal tract. When targeting the intestinal region, additional consideration should be paid to the enzyme's ability to withstand gastric conditions.
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. A well-defined substrate with adequate lot-to-lot uniformity should be used.
7. At a minimum, validation of enzyme assays should document assay specificity, assay variability, assay linearity and assay sensitivity.

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 FDA GMP regulations for dietary supplements. 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, ETA recommends that you consult with your enzyme supplier.

APPENDIX A - ENZYMES FOR DIETARY PURPOSES IN THE U.S. PRIOR TO OCTOBER, 1994*

Enzyme Activity

Enzyme Origin

Alpha-galactosidase

Aspergillus niger

Amylase

Bacillus subtilis

Bacillus amyloliquefaciens

Aspergillus oryzae

Aspergillus niger

Amylase (Malt diastase)

Malt

Amyloglucosidase

Aspergillus niger

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)

Aspergillus oryzae

Bacillus subtilis

Invertase

Saccharomyces cerevisiae

Lactase

Aspergillus oryzae

Kluveromyces lactis

Lipase

Aspergillus niger

Arthrobacter ureafaciens

Candida cylindracea (rugosa)

Rhizomucor miehei

Rhizopus oryzae

Rhizopus delemar

Lysophospholipase

Aspergillus niger

Lysozyme

Egg white

Pancreatin

Porcine pancreas

Pancrelipase

Bovine and porcine pancreas

Pectinase

Aspergillus niger

Aspergillus japonicus

Rhizopus oryzae

Phospholipase

Porcine

Phytase

Aspergillus niger

Protease, microbial

Aspergillus oryzae

Aspergillus niger

Aspergillus melleus

Bacillus licheniformis

Bacillus subtilis

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.