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TOXICITY TESTING

(Micronucleus Assay and Chorioallantoic Membrane Assay)


Toxicity Testing
• Toxicology testing, safety assessment
-conducted to determine the degree to which a substance
can damage a living or non-living organisms
- Identify hazards and risk factors that have the potential to
cause harm (hazard identification)
Toxicity Testing
Categorized by:
• Method of application
• In Vitro
• In Vivo
• Chronicity
• Acute
• Subchronic
• Chronic
• On Route of administration of the toxicant
• Oral
• Dermal
• Inhalation
• Injection (IV, IM, IP, SC)
Toxicity Testing
The major difference between repeated dose and subchronic toxicity
studies is the duration: repeated dose toxicity studies are conducted over
a duration of 28 days, and subchronic toxicity studies are carried out over
90 days. limited exposure of an animal to a substance (acute toxicity) as
well as repeated, long-term exposure (chronic toxicity). Substances are
also tested for more specific endpoints such as cytotoxicity (ability to
damage cells), mutagenicity (ability to cause changes in genetic material),
carcinogenicity (ability to cause cancer), and teratogenicity (ability to
cause birth defects).Identify hazards and risk factors that have the
potential to cause harm (hazard identification)
I. Chemical and Physical Properties
• Probable contaminants from synthesis as well as intermediates
and waste products from synthetic process
II. Exposure and Environmental Fate
A. Degradation studies: hydrolysis, photodegradation, etc.
B. Degradation in soil, water and under various conditions
C. Mobility and dissipation in soil, water, and air
D. Accumulation in plants, aquatic animals, wild terrestial
animals, food plants, and animals
III. In vivo Tests
A. Acute
1. LD50 and LC50- oral dermal or inhaled
2. Eye irritation
3. Dermal irritation
4. Dermal sensitization
B. Subchronic
1.30- to 90-day feeding
2. 30- to 90-day dermal or irritation exposure
C. Chronic/ Reproduction
1. Chronic Feeding (including oncogenicity tests)
2. Teratogenicity
3. Reproduction (multi-generation)
III. In vivo Tests
D. Special Tests
1. Neurotoxicity
2. Potentiation
3. Metabolism
4. Pharmacodynamics
5. Behavior
IV. In Vitro Tests
A. Mutagenicity (Ames Test) – Prokaryote
- Salmonella typhimurium reverse mutation assay
B. Genotoxicity
MICRONUCLEUS ASSAY
INTRODUCTION
Micronucleus Test
– In vivo toxicity study developed by Schmid and co-
workers in 1975.

– Used to screen compounds for clastogenic


(chromosome-breaking) and aneugenic (loss of whole
chromosome activity).
• Micronuclei are extranuclei bioamarker of a
chromosome damage
• One of the standard cytogenetic tools
implemented to assess micronuclei formation
(signifying chromosomal damage) subsequent to
exposure to genotoxic/cytotoxic agents.
• Mutagenic and genotoxic property of the test
substance.
Figure 1: Illustration on formation of micronuclei from damaged chromosomes
(Journal of Radiation and Cancer Research)
Score binucleated cells with
main nuclei that are separate and of
approximately equal size,
main nuclei that touch and even
overlap as long a nuclear boundaries
are able to be distinguished, and
main nuclei that are linked by
nucleoplasmic bridges
Do not score:
trinucleated, quadranucleated, or
multinucleated cells, or
cells where main nuclei are
undergoing apoptosis (because MN
may be gone already or may be
caused by apoptotic process)

The combination of the micronucleus assay with fluorescence in situ


hybridization (FISH)
Micronucleus ( Howell-Jolly bodies)
– chromosomal breakage or spindle damage during
metaphase/anaphase.
– Typically round with as diameter of about 1/20 to 1/5
of the erythrocytes.
– Some are almost almond-shaped.
Principle: Genetic material replicates and divides equally
between two daughter cells.
– Micronuclei are found in a variety of different marrow
cells (myeloblast, myelocytes, erythroblasts,
erythrocytes)
– Majority are found in polychromatic (immature)
erythrocytes
– Giemsa stain: Immature: blue
Mature: pinkish, orange
CHORIOALLANTOIC MEMBRANE
ASSAY
• Screening assay for potential pro-and anti-angiogenic
agents
• An evaluation tool for angiogenesis.
• Assessment of angiogenesis is an indirect assessment of
the carcinogenicity of a drug sample
• Angiogenesis is the formation of new blood vessels and essential in wound healing and
placental formation during pregnancy. It arises in tumors and cancers and may feed and sustain
them and allow cell proliferation and migration. In terms of cancer, tumor angiogenesis is the
consequence of an angiogenic imbalance in which pro-angiogenic factors predominate over
anti-angiogenic factors.
• Alternative versatile, cost effective and ethically unobjectionable in vivomodel for many
applications
• List of feasible applications:
• Angiogenesis (induction, inhibition)
• Tumour cell behaviour (angiogenesis, proliferation, apoptosis, invasion, migration, metastasis)
• Short term tissue culture (tumour samples, small pieces of bone, liver, skin etc)
• Toxicity assay (HET-CAM)
• Testing Biomaterials and drugs
UNTREATED CELECOXIB

DMSO HEXANE

ETHYL ACETATE N-BUTANOL

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