European Toxicology Forum Safety of Food Enzymes

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European Toxicology ForumSafety of Food Enzymes

• William T. Allaben, Ph.D., Fellow, ATS
• National Center for Toxicological Research
• U.S. Food and Drug Administration

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Office of Food Additive SafetyUniversity
Station, River Road, College Park, MD
Home Sweet Home November 2004
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Center for Food Safety and Applied Nutrition
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OFASs Food Ingredients Universe

• Direct food ingredients
• Color additives
• Generally Recognized as Safe (GRAS) ingredients
• Foods/ingredients produced using genetic
  engineering
• Processing Aids
• Food irradiation equipment
• Food packaging/food contact substances

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Overview

• REGULATORY OPTIONS
• Food additive (petition)
• subject to premarket approval
• GRAS determination voluntary notice
• not subject to premarket approval
• SAFETY ASSESSMENT
• Information provided in GRAS notices
• Safety of Food Enzymes
• Enzyme Case Study
• Lipase from a conventional microorganism

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1958 Food Additive Amendment to the Food, Drug
Cosmetic Act

• Defines food additive
• Requires premarket approval of new uses of food
  additives
• Provides for a GRAS exemption
• Establishes the standard of review
• Establishes the standard of safety
• Establishes formal rulemaking procedures for food
  additives

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Regulatory Basis

• In the United States, a food additive is defined
  as
• any substance the intended use of which results
  or may reasonably be expected to result, directly
  or indirectly, in its becoming a component or
  otherwise affecting the characteristics of any
  food if such substance is not generally
  recognized to be safe (GRAS) under the
  conditions of its intended use
• H.R. Report No. 2284, 85th Congress 1958

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REASONABLE CERTAINTY OF NO HARM(Legislative
History of the FDC Act)

• The concept of safety used in this legislation
  involves the question of whether a substance is
  hazardous to the health of man or animal. Safety
  requires proof of a reasonable certainty that no
  harm will result from the proposed use of an
  additive.
• It does not -- and cannot -- require proof beyond
  any possible doubt that no harm will result under
  any conceivable circumstance.

H.R. Report No. 2284, 85th Congress 1958
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U.S. Regulatory Processes for Food Ingredients

• Food Additive Petition
• Regulation in 21 CFR 172 or 173
• GRAS Notice (nearly 200 notices)
• Primary route for submitting information on
  microbial enzymes to FDA
• Information available on the Internet
• www.cfsan.fda.gov/dms/opa-cg7.html

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GRAS Criteria Comparing a GRAS Substance to a
Food Additive
Food Additive
GRAS Substance
Review Approval by FDA
Evidence of Safety
General Recognition
Evidence of Safety
Widely known
Consensus
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The Essence of GRAS

• Premarket approval of food additives unless the
  use of the substance is GRAS
• Information What distinguishes a GRAS substance
  from a food additive
• The information relevant to the safe use of a
  GRAS substance is widely known
• There is consensus among qualified experts that
  the available information establishes that the
  intended use of a GRAS substance is safe
  (case-by-case, e.g. use patterns / chemical /
  toxicological / microbiological )
• Basis for a GRAS determination
• Scientific procedures

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The GRAS ProposalApril 17, 1997

• Proposed notification program for GRAS substances
  
• Notifier prepares summary and tells FDA the basis
  for its GRAS determination
• Three categories of FDA response letters
• No questions regarding GRAS notification
• No basis for GRAS determination
• Withdrawn by notifier

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GRAS Notification Process (1)

• The notifier may ask for a presubmission meeting.
• The notifier elects to send a notice to the FDA.
• States a GRAS exemption claim
• Summary of technical information
• May include report from notifier GRAS Panel
• The FDA sends an acknowledgement letter.

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GRAS Notification Process (2)

• The FDA reviews the notice to determine if it
  provides a basis for the GRAS determination.
• The notifier may withdraw the notice any time.
• After reviewing the notice, FDA sends a letter.
• The FDA maintains a readily accessible file of
  letters and certain information from GRAS notices
  on the Internet
• http//www.cfsan.fda.gov/rdb/opa-gras.html

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Safety of Enzymes Used in Food FDA Perspective

• Animal-Derived Preparations
• Bovine Liver Catalase / Cheeses
• Plant-Derived Preparations
• Barley Produced Amylase / Sugar Syrups
• Microbially-Derived Preparations
• E. Coli K-12 Chymosin / Cheeses

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Microbial Enzyme Submissions

• GRAS Notices - most commonly used means for
  submitting information about microbial enzymes to
  FDA
• Include published and non-published information
• Notices commonly include certain categories of
  information ----

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Information Provided About Microbial Enzyme
Preparations in GRAS Notices

• Safety of the enzyme
• Safety of the production microorganism
• Safety of the manufacturing process
• Safety of the enzyme preparation

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The Enzyme

• Identity
• (name, IUB classification, CAS Reg. No.)
• Enzymatic activity (reactions catalyzed)
• Physico-chemical characteristics
• (e.g., MW, thermal stability, optimum pH)
• Genetic modifications at the DNA and amino acid
  levels (if applicable)

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The Production Microorganism

• Name and taxonomic classification
• Source (animal, plant, microbial)
• Important characteristics
• Nonpathogenic (nor antibiotic properties)
• Nontoxigenic
• History of safe use
• Genetic modifications (e.g. to modify metabolic
  pathways to prevent mycotoxin or proteases
  production)

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For a Bioengineered Production Microorganism (1)

• Characterization of the host strain
• Description of the expression plasmid (genetic
  material, its identity, organization, and origin)

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For a Bioengineered Production Microorganism (2)

• Description of the gene encoding (genetic
  sequence) the enzyme (source(s), modifications)
• Introduction of hereditary materials (DNA) into
  the host and characterization of the production
  strain (no unexpected genetic material
  introduced)

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The Manufacturing Process

• Fermentation (controlled process using food grade
  raw materials)
• Removal of the microorganism
• Recovery of the enzyme
• Purification and concentration
• Formulation and standardization of the enzyme
  preparation

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The Enzyme Preparation

• Consideration of constituents derived from
  source, and manufacturing process
• Use in food
• Level in food (generally very low)
• Estimation of daily intake
• Toxicological studies (case-by-case basis may
  include gene mutation, chromosomal aberrations,
  acute toxicity in rats, etc.)

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The Enzyme Preparation

• Composition (including total organic solids
  TOS)
• Specifications (Food Chemicals Codex, 5th
  edition, 2004 JECFA, 2001)

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Human Exposure to Enzyme Preparations (1)

• Enzyme preparations are used in food at low
  levels (generally at less than 1)
• Exposure for individual enzyme preparations is
  traditionally calculated as TOS (Total Organic
  Solids)

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Human Exposure to Enzyme Preparations (2)

• TOS is the content of organic solids derived from
  the source organism or the manufacturing process
  the major component of TOS is usually the enzyme
  of interest
• TOS () 100 Ash () Water ()
  Formulation aids ()

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Human Exposure to Enzyme Preparations (3)

• Estimated exposure for individual enzyme
  preparations is very low (generally from several
  micrograms to several milligrams TOS/person/day)

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Case Study Lipase

• GRN 000068 Lipase enzyme preparation derived
  from Penicillium camembertii
• Intended Use Production of fatty acids from
  fats and oils
• Similar to other lipases used in food processing
• Source Organism
• Nontoxigenic
• Nonpathogenic
• Long used in the production of Camembert cheese

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Lipase The Manufacturing Process

• Major steps
• Fermentation using food grade ingredients
• Enzyme secreted into medium
• Filtration and heat remove fungal cells
• Ethanol and acid precipitation
• Centrifuged, dried, crushed, blended with
  diluents to desired activity

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Lipase The Enzyme Preparation

• Specifications
• Complies with general and additional requirements
  of Food Chemicals Codex, 4th edition
• Does not contain detectable antibiotics or
  mycotoxins
• Estimated intake 1 mg/person/day

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Lipase Studies

• Pathogenicity study organism (P. camembertii)
  injected into mice no effects, not recovered)
• Toxicological studies using concentrated lipase
  enzyme preparation
• 90 day gavage study in rats up to 2000 mg/kg/d
  no effect
• Bacterial mutagenicity no effects

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Lipase FDA letter

• Based on the information provided by Amano
  Enzyme, Inc., as well as other information
  available to FDA, the agency has no questions at
  this time regarding Amanos conclusion that the
  lipase enzyme preparation derived from P.
  camembertii is GRAS under the intended conditions
  of use. The agency has not, however, made its own
  determination regarding the GRAS status of the
  subject use of this lipase preparation. As
  always, it is the continuing responsibility of
  Amano to ensure that food ingredients that the
  firm markets are safe, and are otherwise in
  compliance with all applicable legal and
  regulatory requirements.

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Summary

• In the U.S.A., microbial enzymes intended for use
  in food are most commonly submitted to FDA as the
  subjects of GRAS notices, which do not require
  premarket review.
• GRAS Notices commonly contain published and
  unpublished information about
• The Safety of the Enzyme
• The Safety of the Production Microorganism
• The Safety of the Manufacturing Process
• The Safety of the Enzyme Preparation

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CFSANs Web Sites of Interest

• Enzyme Preparations
• How to Submit a GRAS
• Summary of all GRAS Notices GRAS
• Notification Program FAQ about GRAS

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Thank You

• Special Thanks to the following CFSAN folks

• Alan Rulis
• Laura M. Tarantino
• Antonia Mattia
• Paulette Gaynor
• Zofia S. Olempska-Beer
• Robert Merker

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CFSAN HOMEPAGE

• www.cfsan.fda.gov

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CFSANs Web Sites of Interest

• CFSAN / OFAS / Enzyme Preparations
  www.cfsan.fda.gov/dms/opa-cg7.html
• How to Submit a GRAS www.cfsan.fda.gov/dms/opa-f
  rgr.html
• Summary of all GRAS Notices www.cfsan.fda.gov/rdb
  /opa-gras.html
• GRAS Notification Program www.cfsan.fda.gov/dms/o
  pa-noti.html
• FAQ about GRAS www.cfsan.fda.gov/dms/grasguid.htm
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