Food Toxicology

Learning Objectives
• Understand the background and principles of
safety assessment of food additives.
• Review the quantitative assessment of dietary
exposure to food additives and the development
Food Additive Safety Assessment of concern levels.
• Explore the basics of testing
related to food additive safety.
Food Toxicology • Explore the relationship between
Instructor: Gregory Möller, Ph.D. food type & food packaging in
University of Idaho food additive safety assessment.
• Understand the interaction of
the Delaney Clause and food
additive regulation.
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Food Toxicology Food Toxicology

Food Additive Scope of Food Additives

• A substance which may, by its intended use, • ~3000 additives
become a component of food, either directly or • ~1800 flavoring agents
indirectly, or which may otherwise affect the • Texturing agents used in highest quantity
characteristics of food.
• Soft drinks are biggest market
– Includes any substance intended
for use in producing, manufacturing, • 4 direct food additives = 93% of total
processing, preparing, treating, – Sucrose, salt,
packaging, transporting or holding corn syrup, dextrose
food, and any source of radiation
intended for such use.

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Food Toxicology Food Toxicology

Six Categories of Food Additives Consumption of Food Additives

• Preservatives • Texture • 139 lbs/year/person
– Antimcrobial – Emulsifiers • 5 lbs/year if remove common ones
– Antibrowning – Stabilizers – Spices, sugars, salt, honey, pepper, mustard, dextrose, etc
– Antioxidant • Miscellaneous • 75% people concerned about food additives
• Nutritional – Enzymes • 60% try to avoid
– Vitamins/minerals – Catalysts
• 6% could name one
• Flavor – Solvents
– Flavor enhancers – Propellants
– Sweeteners
– Nat/syn flavors
• Color
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Consumption of Color Additives Food Additives Numbering Systems

• Estimated max = 53 mg/da • E System
• Estimated average = 15 mg/da – Developed by the European Economic Community (EEC)
• About 10% of foods contain food coloring • Number and description
– e.g. “E123 Amaranth, Colour – Red”
• Additives considered safe and allowed between
countries
• Nutrient additives
not included

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Food Toxicology Food Toxicology

Food Additives Numbering Systems Food Additives

• International Numbering System • Direct food additives
• Developed by Codex Alimentarius • Certified color additives
– Commission Committee on Food Additives and • Exempt color additives
Contaminants • Unintentional additives
– Broader than E system – Tolerance based; Action level based
– e.g. “491 Sorbitan Monostearate;
Emulsifiers and Stabilisers;
salts or Esters of Fatty Acids” Food additive NH4HCO3
• Does not imply toxicology
approval
– INS largely E w/o E in number
– 23 functional classes
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Food Toxicology Food Toxicology

Exempt Chemicals GRAS Ingredients

• GRAS (Generally Recognized as Safe) • Not food additives
– From scientific studies or wide usage • Exempt for pre-market clearance but must be
– Salt, vitamins, etc. supported by safety data
– GRAS - qualified experts determined safe • Exempt from Delaney Clause
– May not be FDA decision (GRAS)
• GRAS list in CFR is not inclusive
– Company can self-proclaim (GRAS)
– Leaves FDA some leeway
• “Prior Sanctioned” before 1958
– Prior sanctioned – approved by
FDA/USDA prior to 1958
– Sodium nitrite, etc.

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GRAS Ingredients: 1997 Changes Regulated Food Additive

• Not all GRAS petitions will be rigorously reviewed • Food additives that are not color, or GRAS,
• Company will submit intention to list as GRAS or prior sanctioned
• FDA may or not review • Require FDA approval
• Speeds process and better directs resources • Scientific data that no harm will occur
– “Redbook” guidelines
• Must justify function
• 32 categories (C&D)
Olestra

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Food Toxicology Food Toxicology

Food Additives Approved Since 1970 Color Additives

• Sucralose acetate isobutyrate 1999 • Same testing as food additives
• Sucralose 1998 • Not eligible as GRAS
• Olestra 1996 • Tested at all FDA concern levels
• Gellan gum 1990 • Two types
• Acesulfame K 1988 – Certified - by FDA chemists for purity
• Polydextrose 1981 – Exempt - mostly naturally occurring

• Aspartame 1981
• TBHQ 1970

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Food Toxicology Food Toxicology

Certified Color Additives Exempt Color Additives

• Prefix of FD&C • Usually naturally occurring (25)
– Two exemptions: Orange C, Citrus Red 2 – Dried algae, beet powder, grape skin extract, fruit juice,
• Every batch must be FDA certified caramel, etc.

• Aromatic amines, aromatic azos • Lack precise chemical identity

– Unusually nontoxic for these compounds • Fade readily, lack intensity and uniformity
• Higher levels required
• Used less (except caramel)

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Contaminant Tolerances Contaminant Action Levels

• For unavoidable contaminants • Informal tolerances
• Acceptable level established • Not legally enforceable
• Any food cannot exceed level • Allow flexibility between different foods
• Enforceable levels (e.g. aflatoxin)
– PCB, metals, Nitrosochemicals • Based on estimated exposures

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Food Toxicology Food Toxicology

Unintentional Food Additives Tests Required

• Packaging materials • FDA Redbook Guidance
– BHA/BHT, PCP, polymers, metals • Toxicological Principles for the Safety
• Processing chemicals Assessment of Direct Food Additives and Color
– PAH (from cooking), solvents (from extractions) Additives Used in Food
• Environmental • What is the safe dose (ADI)?
– Natural and anthropogenic
– Pb, Hg, pesticides, fumigants

Red Book 2000

Click Here
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Food Toxicology Food Toxicology

Definitions Safety Testing of Food Additives

• Reference Dose (RfD): An estimate (with uncertainty • Acute/Short-term = LD50, DRC
spanning perhaps an order of magnitude) of a daily oral • Subchronic - MTD
exposure to the human population (including sensitive
subgroups) that is likely to be without an appreciable risk • Chronic - NOAEL, ADI, RfD
of deleterious effects during a lifetime. • Carcinogenic/Mutagenic
– Can be derived from a NOAEL, LOAEL,
or benchmark dose, with uncertainty • Developmental toxicity
factors generally applied to reflect • Immunotoxicity
limitations of the data used.
• Acceptable Daily Intake (ADI): • Neurotoxicity
The amount of a chemical a person • Metabolic, analytical, etc.
can be exposed to on a daily basis
over an extended period of time
(usually a lifetime) without suffering
deleterious effects.
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Estimating Exposures Direct Food Additives

• Must establish Estimated Daily Intake (EDI) for: • Estimated daily intake
– Direct food and color additives – [Amount of food consumed daily] x [concentration of
– Indirect food additives additive in food]
• The sum of EDIs for an additive from all sources • EDI = concentration (C) x intake (I)
cannot exceed the RfD/ADI • Sum of all sources of additive in food and non-food
• Unique number for each
direct additive

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Food Toxicology Food Toxicology

Direct Food Additives Concentrations in Food

Estimating Consumption Direct Additives
• Level of additive in food category • Determined by manufacturer
• Daily intake of each food category • Assume highest level allowable
containing additive Takeru Kobayashi • Assume processor uses GMP
54 hotdogs
• Distribution of intakes in and does not abuse levels
population groups
• Exposure from non-food
sources

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Food Toxicology Food Toxicology

Regulatory Food Categories (43) Food Categories (43)

• Baked goods and baking mixes • Beverages - nonalcoholic
– Ready to eat and ready to cook products – Special/spiced teas, soft drinks, coffee substitutes,
– Doughnuts, bread, croissants, cake mix, cookie dough fruit/vegetable drinks
• Beverages - alcoholic • Breakfast cereals
– Beer, wine, liquor, cocktail mix – Ready to eat and instant, regular hot cereals
• Of 43 total; see CFR 21, 170.3(n)

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Food Consumption Surveys Food Consumption Survey

Problems
• National Food Consumption Survey
– USDA 1987-8 • 3 – 14 day snapshots
• Continuing Survey of Food Intakes • Lack of detail
by Individuals – Age, ethnic group, food groups
– USDA 1985-91 • Some are outdated – food groups
• Estimates of Daily Intake • Not always “user friendly”
– NAS/NRC 1979
• FDA Total Diet Study 1987

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Food Toxicology Food Toxicology

Estimated Daily Intake Food Safety Concern Levels (CLs)

• Establish EDI • Three levels - CLI, CLII, CLIII
• Assign Concern Level (CL) • Based on “structure-activity” relationships to known
toxic chemicals
• Based on structural and functional groups
– Category A, B, C
• Based on exposure level****

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Food Toxicology Food Toxicology

Toxicity Categories Concern Levels and Exposure Levels

• Category A A B C CL
– Low toxic potential
<0.05 <0.025 0.0125 I
• Category B 0.05 0.025 0.0125 II
– Intermediate or unknown toxicity
1.0 0.5 0.25 III
• Category C
– High degree of toxic concern • Values in ppm (mg/kg) in food
• Much more emphasis on exposure
• Lower exposure - less testing

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Food Additive Safety CLs and Toxicity Testing

Summary CL I
• Estimate daily intake (EDI) • Short-term repeated dose study
• Determine toxicity rating (A, B, C) – Not less than 28 days
– C = higher toxicity • Short-term carcinogen tests
• Assign CL (I, II, III) – Genetic toxicity
– III highest concern, – Ames assay, etc
• Most testing
– Exposure carries
more weight
• CL determines toxicity
tests required
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Food Toxicology Food Toxicology

CLs and Toxicity Testing CLs and Toxicity Testing

CL II CL III
• Subchronic - 90 da - 2 species • Carcinogen studies - rats and mice
• Multigeneration (2) reproduction study with • Chronic feeding study - 1yr - may be combined with
teratology phase carcinogen study
• Short-term carcinogen • Chronic study in non-rodent - 1yr
• Multigeneration (2) reproduction
with teratology phase
• Short-term carcinogen study

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Food Toxicology Food Toxicology

Indirect Food Additives

Toxicity Tests Concern Level • Not added directly to food
I II III • May enter food through migration of packaging
Short-term for genetic toxicity X X X materials, holding containers or processing surfaces
Metabolism and pharmacokinetics X X • Cans, cardboard, plastics, glass, etc.
Short-term rodent toxicity X
Subchronic rodent toxicity X X
Subchronic non-rodent toxicity X
Reproduction with teratology X X
One-year non-rodent toxicity X
Rodent carcinogenicity X
Chronic rodent toxicity/carcinogenicity X
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Estimates of Indirect Additive Migration Indirect Additives Extraction Temperatures

Extraction Methods • Different temps based projected processing method
• Solvents simulate food types • Retorted foods
• Aqueous/acidic food - 8% EtOH – 212°, 250°, 275° for 2 hrs and held 238 hr at 120°
• Alcoholic food - 50% EtOH • Refrigerated foods
• Fatty food - corn oil or – Same but held at 70°
synthetic triglyceride • Frozen foods
• Expose solvent to packaging – Same but held 120 hrs
material to extract

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Food Toxicology Food Toxicology

Indirect Food Additives CFs and fTs

Exposure Estimates
• Consumption factor (CF) = amount of US diet that
• Extraction data = how much in food comes into contact with different kinds of holding
• Extraction data is converted to exposure materials (paper, metal, polymer)
estimates using • Food type (fT) = the fraction of food types for which
– Consumption Factors (CF) each packaging material is used (aqueous/acidic,
– Food-type-distribution factors (fT) fatty, OH)

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Food Toxicology Food Toxicology

Consumption Factors Food-Type-Distribution Factor (fT)

Total fraction of diet in contact with container • Estimate of fraction of food types for which different
• Glass 0.08 types of packaging is used
• Metal, polymer coated 0.17 – Aqueous, acidic, alcoholic, fatty

• Metal, uncoated 0.03 • Performed for each package category

– Glass, metal, paper, polymer
• Paper, polymer coated 0.21
• Paper, uncoated 0.10
• Polymer 0.41

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Food Type Distribution Factor (fT) EDI of Indirect Food Additives

AQU ACID OH FAT • EDI =
• Glass 0.08 0.36 0.47 0.09 CF x [(fT aqueous/acidic x ppm in 8% EtOH)
• Metal, polymer coated 0.16 0.35 0.40 0.09 + (fT alcoholic x ppm in 50% EtOH)
• Metal, uncoated 0.54 0.25 0.01 0.20 + (fT fatty x ppm in corn oil)] x 3kg/person/day
• Paper, polymer coated 0.55 0.04 0.01 0.40 = mg/person/day
• Paper, uncoated 0.57 0.01 0.01 0.41 – CF = consumption factor
• Polymer 0.49 0.16 0.01 0.34 – fT = food type distribution
factor
– 3 kg=daily food consumption

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Food Toxicology Food Toxicology

Indirect Additives Indirect Food Additives

EDI Summary Toxicity Testing
• EDI = • Negligible migration (< 0.05ppm) and
(sum of fTs x extraction data in ppm) x EDI < 0.15mg/person/day
3kg/person/day x CF – Acute toxicity studies only
• Total of EDIs cannot exceed ADI • Migration 0.05-1.0ppm
– Subchronic studies; 2 species
• Migration > 1.0ppm
– Chronic studies; 2 species
– Carcinogenicity
– Multigenerational reproduction
– Teratology
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Food Toxicology Food Toxicology

Threshold of Regulation FDA Modernization Act 1997

• New legislation on indirect food additives - 1997 • Indirect food additives can be marketed 120 days
• Concentrations <0.05 ppb and not carcinogenic after notifying FDA
• Exempt from full-blown pre-market evaluation and • Burden of objection on FDA
petition review • Significantly streamlined process
• Assume negligible risk • FDA must review much faster

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Dietary Supplements Delaney Clause and Food Additives

• Regarded as food • Prohibits FDA approval of regulated food
• Not food additives or drugs additives shown to cause cancer by appropriate
• Lesser standard of safety tests
– DS: no history of unsafe use • Appropriate tests are usually animal studies
– Additive: demonstrated safety
• No FDA unapproved
health claims
– Would then be a drug
– Different safety testing

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Food Toxicology Food Toxicology

Delaney Clause FDA Carcinogenic Constituent Policy

For Non-functional Carcinogenic Contaminants of Food Additives
• Applies to:
– Regulated food additives, color additives and drugs • Delaney is not invoked
• Does not apply to: • Health effects may be negligible
– Unavoidable contaminants, GRAS substances, • Probabilistic RA model
prior sanctioned ingredients or non-functional trace
contaminants
Standardized Normal Distribution
Probability

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Carcinogenic Constituent Food Toxicology Food Toxicology

Policy Risk Assessment Model De Minimis Concept

• Upper bound lifetime risk in humans < 1 in 106 above • De minimis non curat lex
background – “The law does not concern itself with trifles”
• Many conservative estimates made in model • First involved insect and worm fragments in food
– Possibly over estimates risk by million-fold – Cannot completely avoid
• Later applied to carcinogenic contaminants in food
additives

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De Minimis Concept Carcinogenic Constituent

Diethylstilbestrol (DES) Proviso
• Most recently courts have not allowed FDA to invoke
de minimis because Delaney language is specific • Permits addition of carcinogenic substances to
• FD&C orange 17 animal feeds if no residues end up in edible
• Pesticide residues tissues
– Resulted in FQPA changes

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Food Toxicology Food Toxicology

Interpretation of Delaney: Food Additives Interpretation of Delaney

• RA cannot be used if food additive is carcinogenic • Must be primary carcinogen
• Zero tolerance policy • Secondary carcinogenesis not considered evidence
• Very strict regulation
• FDA requires clear, unequivocal,
reproducible evidence for cancer
• Few substances banned

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Food Toxicology Food Toxicology

Secondary Carcinogenesis Secondary Carcinogenesis

• Nutritional, hormonal, physiological imbalances are • BHA
secondary causes of cancer – Chronic irritation = tissue damage = hyperplasia = cell
• Secondary carcinogens only contribute to increase proliferation = increased chance for mutation and cancer
(promote) effect • Xylitol and sorbitol
• No evidence of direct genotoxicity – Calcium imbalance in gut
exacerbated by fermentation
of sugar alcohol

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Substances Banned by Delaney Carcinogenic Food Additives

• Packaging materials - Flectol H, mercaptimidazoline • BHA, xylitol, sorbitol, methylene chloride, TCE,
• Food additives - safrole, cinnamyl anthranilate, melamine, formaldehyde, nitrilotriacetic acid,
thiourea, diethylpyrocarbonate (forms urethane) diethylhexyl phthalate, bentonite
• Primary carcinogens • All listed by National Toxicology Program and
International Agency for Regulation of Carcinogens
• All secondary carcinogenesis
– FDA approved

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Food Toxicology

Delaney Clause
• Because of the strict nature of the Delaney Clause,
the FDA requires clear and unequivocal proof that an
additive is a carcinogen

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