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Wongwiwat Tassaneeyakul
Department of Toxicology
Khon Kaen University
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 To understand the important role
of toxicity test in drug/chemical
development,
 Can describe the component of
toxicity test commonly used,
 Understand the relationship
between these tests.
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~100 Discovery Approaches
High Risk Process:
11-15 Years, $800MM+
Millions of
Compounds Screened
Preclinical
Pharmacology
Preclinical Safety
1-2
Products
Clinical Pharmacology
& Safety
Discovery
Exploratory Development
Phase I
0
Idea
Full Development
Phase II
Phase III
15
10
5
11 - 15 Years
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Drug
3
Frame work for Systems Toxicology
Waters & Fostel, 2004
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Procedures on Living Animals :
Great Britain 2000

Total number of Procedures
 Total number of animals used

2,714,726
2,642,993
Total number of toxicology procedures 454,904
(16.8%)
• Total number of acute lethal tests in the rat (LD50/LC50)


2292
Total number of Procedures in Dogs
7,632 (0.3%)
Total number of procedures in primates
3,690 (0.1%)
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Toxicity Tests
1.
2.
3.
4.
Acute toxicity test (single dose)
Subchronic toxicity test (repeated dose)
Chronic toxicity test (repeated dose)
Special test
4.1 Mutagenicity test
4.2 Development and reproductive test
4.3 Carcinogenic test
4.4 Neurotoxicity,
4.5 etc………………….
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Animals Toxicity Tests
Assumption
1) Extrapolation from animal to human
is possible
1.1 Appropriate dosage adjustment
1.2 Known human carcinogen are
carcinogenic in (some) animals
1.3 Species differences mostly due
to different metabolic pathways
2) High dose exposure is a necessary
(to reduce a less number of animal
used)
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Acute Toxicity Test

It involves lethal dose/concentrations and short-term
exposures,

Appear immediately after exposure,

The end point is usually death, hence it is used to derive
LD50 /LC50

An LD50 is a dose of a toxic chemical that kills half of the
population.

LD50 is obtained by plotting, for a given dose the
proportion of the population that responded to that dose
and all lower doses
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Acute Toxicity Tests
OBJECTIVES
1. Estimate LD50 or LC50 for
comparison
2. Identify target organ of intoxication
to predict toxicity effect in human
3. Establish reversibility of toxicity
4. Calculate dose range guiding for
further repeated-dose test
COMPONENTS
Acute lethality + Eye irritation + Skin test
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Acute Lethality Tests
(LD50 test)
METHOD
Route: intended route (e.g. p.o. or parenteral)
Species: 1 rodent + 1 non-rodent
Dose : > 5 level
Observed period: up to 14 days
INDICATORS
LD50 + 95% confidence interval
Functional toxicity
Histo/pathology, hematology, autopsy, etc.
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Alternative acute toxicity tests
1. Approximate lethal dose (ALD) “1.5”
2. Up and down method
3. The British Society of Toxicology (BST)
protocol
5, 50, 500 mg/kg then up and down adjust
4. The “Fixed Dose” procedure
5, 50, 500, 2000 mg/kg
very toxic, toxic, harmful, nontoxic
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Up and down method
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Aquatic toxicity testing
Daphnia (water flea)
Daphnia toxicity test
“White rat of aquatic
toxicity testing”
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Example problem
Dose
0 mg/L (control)
1
3
10
30
100
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% alive
100
100
90
30
20
0
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Eye Irritation (Draize) Test
METHOD
Exception of test : pH <2 or >12
Route: eye
Species: Rabbit (New Zealand White)
Dose : 0.01- 0.1 ml or 100 mg
Control : contralateral eye
Measurement : cornea, iris, conjunctiva
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Skin Irritation Test
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Skin Sensitization Test
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Subchronic Toxicity Tests
OBJECTIVES
1. Estimate NOAEL (No observed
adverse effect level), MTD (maximal
tolerable dose)
2. Identify target organ of intoxication
after repeated dose exposure
3. Calculate dose range guiding for
chronic toxicity test
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MTD = highest dose that suppress <10% body wt gain in
90-days when compare to control group.
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Subchronic Toxicity Tests
METHOD
Route: intended route
Species: 1 rodent + 1 non-rodent
Dose : > 3 level + control
high dose ………….. < 10% fatality
…………
low dose ……………. No apparent toxicity
Observed period: 30-90 days
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Subchronic Toxicity Tests
MEASUREMENT
1) Interim test : to establish baseline of
appearance
food consumption, body wt
hematological/ biochemical test
urinanalysi
2) Termination test : as interim
+ histopathological /autopsy
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Chronic Toxicity Tests
It involves Sub-lethal concentration and longterm exposure,
Effect could be anything (biochemical,
physiological), but not death,
Chronic toxic response can last a long time
or be permanent,
 Under chronic conditions the organism
survives but production or gene frequency
could be affected,
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Chronic Toxicity Tests
OBJECTIVES
1. Identify the spectrum of toxicity over a
broad range of dose
2. Extrapolation of adverse effect to
human
3. Prediction of safe level of exposure in
human (Safety Factor, SF)
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Chronic Toxicity Tests
METHOD
Route: intended route
Species: 1 rodent + 1 non-rodent
Dose : > 3 level + control
high dose ………….. MTD
then 1/4, 1/8, ………..
Observed period: >90 days to 2 yrs
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Development and Reproductive
Toxicity Test (DART)
Objective:
To evaluate potential toxicity on
developmental animals and
reproductive system.
Study Type
Multigenerational (2-3 G) study
Three-segments single
generation study
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Embryonic development and critical period for
teratogenesis
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Possible Targets in DART
Reproductive system
Key hormones
Spermatogenesis/ Ovulation
Mating function
Sperm viability/ Fertilization
Placentation
Maternal behavior
Developing offspring
Implantation development
Organ development & maturation
Embryo / fetal growth
Suckling behavior
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Indices used in DART
1. Fertility index
= # females pregnant
# females paired
2. Implantation index
= # implantation sites
# corpora lutea
3. Gestational (live birth) index
= # lived pups born
# pups delivered
4. Viability index
= # pups surviving 4 days
# lived pups at birth
5. Sex ratio
= # male offspring X 100
# female offspring
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(A) Litter size after
exposure to herbicide
mixture; summary of
groups (n = 275).
Distribution of litter
size in each dosing
group: (B) control (n =
62); (C) very low dose
(n = 31); (D) low dose
(n = 58); (E)
intermediate dose (n
= 61); (F) high dose (n
= 63). Interm,
intermediate.
*p < 0.05, ANOVA,
Bonferroni.
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Multigenerational study
F0 mating
F1B mating
F1A
autopsy
F2A
autopsy
F3A
autopsy
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F2B mating
F3B
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DNA REPAIR MECHANISMS
SHORT-TERM CONSEQUENCES
PHYSIOLOGICAL
DYSFUNCTION
CELL DEATH
Decreased
Genomic
cellular
instability
proliferation
ABNORMAL GROWTH &
METABOLISM
Impaired
protein/ gene
expression
Defective
signalling
pathways
LONG-TERM CONSEQUENCES
Ageing
Cancer
Disease
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Test of Genetic
Damage
DNA DAMAGE
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The Ames/Salmonella Test
“I know, let’s invent
the Ames test”
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Prototrophic Bacteria Cell (His+/+)
Energy Source
1
3
B
A
C
5
D
2
7
E
4
F
G
Histidine
+ Other amino acids
Protein synthesis
6
Cell Growth
Waste Product
Auxotrophic Bacteria Cell (His-/-)
Energy
Source
1
A
3
B
C
2
Waste Product
5
7
No histidine+ Other amino acids
D
4
Protein synthesis decrease
6
No Cell Growth
Mutation in enzyme 4
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http://faculty.washington.edu/jclara/301/M301lecOut/MuRepair.html
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