Toxicity Test

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Toxicity Test
Wongwiwat Tassaneeyakul Department of
Toxicology Khon Kaen University
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Objective of Learning

• 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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High Risk Process 11-15 Years, 800MM
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Frame work for Systems Toxicology
Waters Fostel, 2004
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Procedures on Living Animals Great Britain 2000

• Total number of Procedures 2,714,726
• Total number of animals used 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

• 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

• Estimate LD50 or LC50 for comparison
• Identify target organ of intoxication to predict
  toxicity effect in human
• Establish reversibility of toxicity
• 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 gt 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

• Approximate lethal dose (ALD) 1.5
• Up and down method
• 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) White rat of aquatic
toxicity testing
Daphnia toxicity test
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Example problem

• Dose alive
• 0 mg/L (control) 100
• 1 100
• 3 90
• 10 30
• 30 20
• 100 0

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Eye Irritation (Draize) Test
METHOD
Exception of test pH lt2 or gt12 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

• Estimate NOAEL (No observed adverse effect
  level), MTD (maximal tolerable dose)
• Identify target organ of intoxication after
  repeated dose exposure
• Calculate dose range guiding for chronic toxicity
  test

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NOAEL , LOAEL, MTD
MTD highest dose that suppress lt10 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 gt 3 level control high dose
.. lt 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
  long-term 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

• Identify the spectrum of toxicity over a broad
  range of dose
• Extrapolation of adverse effect to human
• 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 gt 3 level control high dose
.. MTD then 1/4, 1/8, .. Observed
period gt90 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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Phocomelia
THALIDOMIDE
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Possible Targets in DART
Reproductive system Key hormones Spermatogenesi
s/ 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 lt 0.05, ANOVA, Bonferroni.
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Multigenerational study
F0 mating
F1B mating
F1A
autopsy
F2B mating
F2A
autopsy
F3B
F3A
autopsy
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DNA DAMAGE
DNA REPAIR MECHANISMS
SHORT-TERM CONSEQUENCES
Test of Genetic Damage
ABNORMAL GROWTH METABOLISM
PHYSIOLOGICAL DYSFUNCTION
CELL DEATH
Decreased cellular proliferation
Impaired protein/ gene expression
Genomic instability
Defective signalling pathways
LONG-TERM CONSEQUENCES
Cancer
Disease
Ageing
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The Ames/Salmonella Test
I know, lets invent the Ames test
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http//faculty.washington.edu/jclara/301/M301lecOu
t/MuRepair.html
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