Lecture 13

Question 1

Why toxicity testing?

Answer 1

Thalidomide/Contergan disaster

Question 2

What do toxicity tests depend on?

Answer 2

• Type of substance
• Regulations of particular countries
• Use of the test agent

Question 3

What are some of the biological considerations in toxicity testing?

Answer 3

• Most appropriate test species or strain
• Sex of test animal Metabolic similarity to human body
• Physiological similarity to human target tissues
• Modulatory effects due to immune response
• Route of administration and vehicle
• Diet of test animals
• Duration of toxicity testing
• Number of test animals

Question 4

What are some physio-chemical properties of test substance?

Answer 4

• Structure (chemistry of functional groups)
• Vapour pressure (equilibrium pressure of a vapor above its liquid)
• pKa (acid dissociation constant)
• Solubility (water vs fat) Melting point Purity

Question 5

LD50?

Answer 5

Median lethal dose - Dosage causing 50% mortality in exposed animals

Question 6

Describe 1-6 Toxity Ratings

Answer 6

1. Super Toxic <5
2. Extremely toxic 5-50 range
3. Very toxic 50-500 range
4. Moderately toxic 500-5000
5. Slightly Toxic 5000-15,000 range
6. Practically non-toxic

Question 7

Why may LD50 values vary?

Answer 7

Test results may vary considerably between laboratories due to:
* Differing genotype or phenotype of sample population
* Difference in tested range of animal species
* Influence of environmental factors
* Mode of drug adminstration

Question 8

Name and describe a slightly toxic Agent

Answer 8

Ethanol (high LD50 value)
* Slightly toxic, acte effects probably based mostly on non-specific membrane pertubations, chronic exposure triggers side effects in various tissues based on drastic metabolic disturbances
* Ethanol consumption exeeds that of any other drug or mild toxin
* Major effect through non-specific perturbation of membrane signaling mechanisms, but pathophysiological effect of ethanol is mediated also via binding to GABAAR [ϒaminobutyric acid type A ]- type inhibitory ligand-gated ion channels in CNS

Question 9

Name and describe a super toxic agent

Answer 9

Botulinum Toxin (extremely low LD50 value)
* Extrememly potent neurotixin with highly specific pathophysiolocal effects
* Produced by the anaerobic bacillus Clostridium botulinum
* Cause of severe cases of food poisoning (botulism)
* Botulinum toxin is a protein that tightly binds to cholinergic nerve terminals
* Botulinum toxin inhibits specifically the release of the neurotransmitter ACh by degrading key proteins for the vesicular docking process

Question 10

Why is the LD50 dose-response curve helpful?

Answer 10

• Helpful to determine the concentration of an agent that causes the median lethal dose

See slide 14-15

Question 11

Name the tranditional whole animal toxicity tests itilized in the toxicological risk assessment process

Answer 11

• Acute toxicity tests (single exposure)
• Sub-chronic toxicity tests (repeated exposure)
• Chronic toxicity tests (repeated exposure)
• Reproductive toxicity tests
• Teratogenicity (developmental) toxicity tests
• Ocular/skin toxicity tests
• Hypersensitivity tests
• Toxicokinetic tests
• Behavioural tests

Question 12

Characteristics of a proper “in vitro” test system.
(For screening purposes)

Answer 12

• Good correlation with in vivo toxicity data
• Low incidence of false positives
• Low incidence of false negatives
• Good economic profile: Inexpensive and fast

Question 13

Characteristcs of a proper “in vitro” test system
For regulatory purposes

Answer 13

• High interpretability
• Mechanistically based assay
• High predicitve power
• Rapid and reliable tests

see slide 20 for advantages of a proper “in vitro” test system

Question 14

Name the types of in vitro test systems

Answer 14

Cell culture-based cytotoxicity assays
* Kenacid Blue total protein assay
* MTT (methyl tetrazolium) metabolism assay
* Neutral Red dye uptake assay
* Crystal Violet dye assay
* ATP content assay
* LDH leakage assay
* Chicken egg CAM test
* Genotoxicity AMES test (Salmonella)
* Organ/Tissue-derived cell testing
* Organ on a Chip

Question 15

Steps in Neutral Red dye uptake assay

Answer 15

• Seed cells in 96-well plates
• Dose plates with different concentrations of test substance
• 1 to 24 hour incubation time
• Addition of analytical reagen, e.g Neutral Red dye
• 3 hour incubation
• Washing step
• Analysis of dye uptabe by cells. Read at wavelength=550nm
• Only living cells store Neutral Red
• Amound of dye retained by cultured cells is an indication of the number of livings cells.
• Loss of neutral red uptake = loss of cell viability
• Calculation of IC50 value

Question 16

What can a Genotoxicty AMES test be used for?

Answer 16

• AMES test can be used for the initial screening of potential genotoxins
• AMES test determines mutagenicity of test chemicals prior to measuring mutagenic potential in animals

Question 17

Genotoxicity AMES test (Salmonella)

Answer 17

• Histidine - requiring Salmonella strain
• Inoculation of a plate containing histidine-lacking medium
• Histidine requiring bacteria will NOT grow in the medium
• Potential mutagenic chemical is placed centrally in a well in the agar plate
• Diffusion of test substance
• Incubation of his- Salmonella strain
• If the chemical is not a mutagen, colonies fail to appear
• If the chemical is a mutagen, some bacteria will undergo a mutation
• If bacteria can synthesize histidine, colonies will appear

Question 18

What does Chicken Egg HET-CAM Test assess?

Answer 18

HET (Hen’s egg test) – CAM (chorioallantoic membrane) assay to assess membrane irritation

Question 19

Procedure of Chichen Egg HET-CAM Test

Answer 19

• Chicken egg
• Removal of part of the shell of fertilized egg
• Exposure of delicate veined choriallantoic membrane
• Application of test chemical directly to membrane (surrounded by Teflon ring)
• Incubation (minutes to hours)
• Analysis of potential membrane irritation

Question 20

What do cell-based toxicty tests exist for?

Answer 20

• Liver
• Heart
• Kidney
• Blood
• Lungs
• Placenta
• Nervous System