Quiz

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Quiz

• What are the two major factors considered in
  determining the safety of a food additive?

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Toxicity Testing
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(No Transcript)
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Acute Toxicity

• PURPOSE
• To determine the median lethal dose (LD50) and to
  provide observations for subsequent studies.
• ANIMALS
• At least two species, rats and mice are the most
  common.
• At least 10 animals, 5 per sex,
• DOSAGE
• Single administration by gavage or capsule or
  multiple administration over a 24 hour period
• At least 3 dose levels bracketing the range from
  10 to 90 mortality.
• Controls generally not required.

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Acute Toxicity

• OBSERVATIONS
• Animals observed twice daily for 14 days.
• Observations recorded individually for each
  animal should include time of onset, duration,
  and intensity of toxicological and
  pharmacological signs. complete gross necropsy on
  all dead animals.
• INFORMATION
• LD50
• Guide lines for doses to be used in sub-acute
  testing
• Nature of potential toxicity
• Target organs

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SHORT-TERM (28 DAY) CONTINUOUSEXPOSURE ORAL
TOXICITY TESTS

• PURPOSE
• To determine target organs and establish dosages
  for sub-chronic studies.
• ANIMALS
• At least two species, rats and dogs most common.
• At least 10 animals/sex for rats and 4
  animals/sex for dogs for each dose.
• DOSAGE
• Continuous exposure.
• At least three, preferably five, dose levels with
  the highest level causing significant toxicity
  and the lowest showing no toxicity.
• Controls for diet and carrier when the carrier
  has not been well characterized.

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SHORT-TERM (28 DAY) CONTINUOUSEXPOSURE ORAL
TOXICITY TESTS

• OBSERVATIONS
• Daily observations similar to those described for
  the acute toxicity studies
• Hematology to include hematocrit, cell counts,
  and clotting time
• Complete blood chemistry at end of test period to
  include serum enzymes
• Gross necropsy including organ weights.
• HISTOPATHOLOGY
• Non rodents-all gross lesions, heart, ovaries,
  and spleen. All animals are examined.
• Rodents-all gross lesions.
• In addition for animals in the control and high
  dose groups heart, lung, thyroid, parathyroid,
  stomach, small large intestine, uterus, brain,
  lymph node, testes, ovaries, spleen and bone
  marrow are examined. Other groups are examined if
  changes or equivocal results are seen.
• INFORMATION
• Affected organs Dosages for subsequent experiments

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SUB-CHRONIC (90-360 DAYS) ORAL TOXICITY

• PURPOSE
• Characterize the toxicity of a substance and to
  define a level that produces "no observed adverse
  effects." NOEL
• ANIMALS
• Similar to short-term tests except that twice the
  number of animals are used.
• DOSAGE
• Continuous exposure for at least 90 days.
• OBSERVATIONS
• Daily observations.
• Tests similar to short-term tests with the
  addition of some additional organs.

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METABOLIC PHARMACOKINETIC STUDIES

• IN VITRO
• Effects of enzymes and gut flora on compound
• Fate of compound in foods
• IN VIVO
• Absorption
• Distribution in tissues
• Metabolic fate
• Excretion
• Changes in enzyme profiles
• Time course of A to E
• Dose response of A to E

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Mutagenicity

• IN VITRO
• Ames test
• Cultured cells with activating system
• Induction of unscheduled DNA synthesis or repair
• INDIRECT IN VIVO
• Host mediated assay
• Exposure of cultured cells or organisms to body
  fluids
• IN VIVO MUTAGENESIS
• Chromosomal changes at metaphase
• Micronucleus
• Dominant lethal

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Reproductive Effects

• At least 2 species
• At least 2 doses (1 toxic control)
• 3 Generations with 2 litters/generation
• Treat for 60 days prior to breeding and then
  throughout lifetime of offspring
• Observations
• Fertility Length of gestation
• Live births Still births
• Survival at 4 days
• Survival at weaning
• Body weights
• Gross abnormalities
• Teratogenicity
• Birth defects

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Long Term Toxicity

• PURPOSE
• A broad screen for toxicity that will define
  effects, establish the NOEL, and determine
  carcinogenicity
• ANIMALS
• Rodents-50/sex/group with increases for
  sacrifices planned before the end of the test.
• Dogs-8/sex/group
• DOSAGE
• At least three plus control.
• Lowest dose should not show toxicity.
• At least 50 of the animals should survive for 24
  months.
• The highest dose is usually the highest amount
  that can be administered without reducing the
  life span of the animals unless that dose exceeds
  5 of the diet.

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Long Term Toxicity

• OBSERVATIONS
• Daily observations similar to sub-chronic
  studies.
• Histopathology to include
• Adrenal Peripheral Nerve
• Aorta Pituitary
• Bone Prostate
• Bone Marrow Rectum
• Brain (3Levels) Rep. Lymph Nodes
• Caecum Salivary Glands
• Colon Seminal Vesicle
• Corpus Cervis Uteri Skeletal Muscle
• Duodenum Smooth Muscle
• Esophagus SpinalCord (2Levels)

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Long Term Toxicity

• Eyes Spleen
• Gall Bladder Sternum
• Heart Stomach
• Ileum Testes
• Jejunum Thymus
• Kidneys Thyroid (Parathyroid)
• Liver Trachea
• Lungs Urinary Bladder
• Mammary Glands All tissues showing abnormality
• Ovaries  
• Pancreas

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Problems Associated with Determining the Safety
of Food Additives

• Absolute safety cannot be proven
• No protocol is adequate for all compounds
• Extrapolation from high doses to low dose
• Extrapolation from animals to man
• Genetic variation
• High cost
• Additives may be safer than the food itself

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EXTRAPOLATIONS

• IN DIET ANIMALS W/CANCER
• 1.0 10.
• 0.1 1.0
• 0.01 0.1
• 0.001 0.01
• 0.0001 0.001
• At each level, how many animals do you need to
  end up with 1 animal having tumors?

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SOME IMPORTANT CONSIDERATIONS IN TOXICITY
EVAULATION

• COMPLICATING FACTORS
• Storage in tissues
• Conversion to
• More toxic compounds
• Carcinogenic compounds
• Toxic impurities
• Synergism or antagonism
• Genetic differences
• Promoters

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SOME IMPORTANT CONSIDERATIONS IN TOXICITY
EVAULATION

• SIMPLIFYING FACTORS
• Non-absorption
• Not metabolized
• Excreted
• Conversion to
• Normal metabolic species
• Less toxic forms

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Risk Assessment

• Definition quantification of exposure
• Characterization of the exposed populations in
  quantitative terms
• Chemical physical properties of the substance
  and its chemical reactivity in relation to
  exposure
• Prudent mathematical extrapolation of the
  responses from observed to estimated exposure
  ranges
• Qualification of the estimated risk in light of
  identifiable biologic and toxicologic differences
  in the human population