Quiz

1. Quiz What are the two major factors considered in determining the safety of a food additive?

2. Toxicity Testing

4. 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.

5. 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

6. 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.

7. 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 organsDosages for subsequent experiments

8. 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.

9. 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

10. 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

11. 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

12. 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.

13. Long Term Toxicity OBSERVATIONS: Daily observations similar to sub-chronic studies. Histopathology to include: AdrenalPeripheral Nerve AortaPituitary BoneProstate Bone MarrowRectum Brain (3+Levels)Rep. Lymph Nodes CaecumSalivary Glands ColonSeminal Vesicle Corpus & Cervis UteriSkeletal Muscle DuodenumSmooth Muscle EsophagusSpinalCord (2+Levels)

14. Long Term Toxicity EyesSpleen Gall BladderSternum HeartStomach IleumTestes JejunumThymus KidneysThyroid (Parathyroid) LiverTrachea LungsUrinary Bladder Mammary GlandsAll tissues showing abnormality Ovaries Pancreas

15. 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

16. EXTRAPOLATIONS % IN DIET% ANIMALS W/CANCER 1.010. 0.11.0 0.010.1 0.0010.01 0.00010.001 At each level, how many animals do you need to end up with 1 animal having tumors?

17. 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

18. SOME IMPORTANT CONSIDERATIONS IN TOXICITY EVAULATION SIMPLIFYING FACTORS: Non-absorption Not metabolized Excreted Conversion to: Normal metabolic species Less toxic forms

19. 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