pengenalan kepada toksikologi

1. General Toxicology
Dr. Noor Rain Abdullah
Herbal Medicine Research Center
Institute for Medical Research,
Kuala Lumpur

2. General Toxicity testing referred to a series of toxicity
testing required by International regulatory for
compliance to Good Laboratory Practices (GLP) for
proof of safety in experimental animal prior to their
testing in human. .
It comprises of :
Acute,
Sub-acute
chronic toxicity.
These studies are conducted based on “OECD guidelines
of testing of chemicals”
They are performed in rodents and non rodents

3. International regulations relating to human health require
that all new pharmaceutical drugs undergo Preclinical
investigation.
One of the phases of the Preclinical investigation is the
toxicology studies,
It is conducted following an appropriate guideline and
quality system (OECD GLP).

4. Preclinical Studies
Includes:
1. Phytochemistry , Development of the
products
2. Formulation
3. Complete pharmacologic and
pharmacokinetic profile
4. Safety study – Toxicology
5. Efficacy study

5. Regulatory Guidelines and
Quality System in Toxicology
drug safety testing is driven by
International and national
regulatory requirements, e.g.
OECD/ICH guidelines, Good
Laboratory Practices (GLP)….

6. GLP
GLP is a quality system
concerned with the organ
izational process and the
conditions under which n
on-clinical health and env
ironmental safety studies
are :
PLANNED

7. performed,
monitored,
.
GLP

8. recorded, archived and reported

9. Preclinical Toxicology
In vitro Studies
Genotoxicity test (to predict
carcinogenicity)
• Ames (Bacterial Reverse
Mutation Test ) testing to
measure effects on single
DNA bases ,
• the micronucleus assay
to measure structural or
numerical changes to
chromosomes
• Chromosomal abberation
• Mouse Lymphoma
In vivo studies: Animal
testing
• 􀂊 Efficacy
• 􀂊 Toxicity
Acute
Subacute
Chronic

10. General Toxicology
According to the OECD Guidelines
1. Test Substance
2. Experimental Animals and maintenance
Animal selection,
Species and strain
selection,
Age and Weight,
Husbandry
4. Parameters measured
During the period of test
5. End of Study
6. Data Analysis
7. Data Reporting
Dose level,
Dose selection,
Administration of
test substances
3. Treatment Design
Clinical observation,
Body weight, food intake,
and water intake
Clinical Pathology,
• Hematology,
•Clinical Biochemistry
•Gross necropsyThe evaluation of results will
be with respects to the following:
Clinical observation, Body
weight, food intake, water inyake,
Hematology, Clinical Chemistry,
Gross necropsy, Histopathology
Statistically

11. Acute Toxicity
Definition :
Acute toxicity is toxicity study aim to determine acute
effect of chemical produced after administration of a
single dose (or multiple doses) to experimental animal in
a period not exceeding 24 hours,
Dose depending on the guideline can be up to a limit of
2000 and 5 000 mg/kg BW
Objective of acute toxicity studies: To identify a dose
causing major adverse effects and an estimation of the
minimum dose causing lethality, according to regulatory
guidelines and information, guide on doses for the sub-
acute test

12. Acute Toxicology
Rats, female
Study group
Dose level and selection
Admission of test Subs, orally
Findings
OECD Guidelines 420, 425
5 rats
Control
5 rats
5 g/kg
Group 1 Group 2

13. OECD Guidelines for the Testing of Chemicals
Test No. 420: Acute Oral Toxicity - Fixed Dose
Procedure
Principle : in the main study only moderately toxic
doses are used, and the administration of doses that
are expected to be lethal should be avoided.
Sighting study : Groups of animals of a single sex
(normally females) are dosed in a stepwise
procedure using the fixed doses of 5, 50, 300 and
2000 mg/kg (exceptionally 5000 mg/kg). If dose
causes acute effect, stop at that dose, if not cont
until the highest dose.
Main study : Two groups of 5 rats each, one control
and one test

14. OECD Guidelines for the Testing of Chemicals
Test No. 425: Acute Oral Toxicity: Up-and-Down
Procedure
Principle :to apply to materials that produce death
within couple of days.
Sighting study : step wise
Main study : Two groups of 5 rats each, one control
and one test

15. Acute Toxicity (OECD Guideline 420, 425)Acute Toxicity (OECD Guideline 420, 425)
Parameters:
Mortality, Clinical pathology, Gross necropsy, Weight change,
Clinical observations
Points to consider:
Dose selection for repeat dose studies
Objective: To determine
Maximum Tolerated Dose (MTD)
No Observable Effect Level
(NOEL)
To help select doses for
repeated-dose study,
Duration: 14 days after single
dose
Animals Required: (rodent)

16. What is subacute toxicity
• The effect of exposure from 1 day to 30
days

17. OECD Guidelines for the Testing of Chemicals
Test No. 407: Repeated Dose 28-day Oral Toxicity
Study in Rodents
Principle : Provides information on health hazard likely to arise from
exposure (daily) to test substance via oral administration.
The method is based on the repeated oral administration of the
substance of interest during one limited period (one dose level daily
during 28 days).
This Guideline is intended primarily for use with rodents (rat
preferably). At least 10 animals (5 female and 5 male) should be
used for each dose level.
Three tests groups, should be used.
The test compound is administered by gavage or via the diet or
drinking water. A limit test may be performed if no effects would be
expected at a dose of 1000 mg/kg bw/d.

18. Subacute (Guidelines 407)
SD Rats, male and Female
5 rats
5 female
5 rats
5 female
5 Rats
5 female
5 rats,
5 female
Control Low Dose Medium Dose High Dose
Study group
Dose level and selection
Admission of test Subs,
oral
Group 1 Group 2 Group 3 Group 4
Findings

19. What is chronic toxicity
• Daily exposure for 3 months or more

20. OECD Guidelines for the Testing of Chemicals
Test No. 452: Chronic Toxicity Studies
The objective : of these chronic toxicity studies is to characterize the
profile of a substance in a mammalian species (primarily rodents)
following prolonged and repeated exposure.
The Test Guideline focuses on rodents and oral administration. Both
sexes should be used. For rodents, at least 20 animals per sex per
group should normally be used at each dose level, while for non-
rodents a minimum of 4 per sex per group is recommended.
At least three dose levels should be used in addition to the concurrent
control group.
Frequency of exposure normally is daily, but may vary according to the
route chosen (oral, dermal or inhalation) and should be adjusted
according to the toxicokinetic profile of the test substance. 12
months or longer or or up to 10% of species’ lifespan. Length
depends on intended period of human exposure

21. Chronic (Guidelines 452)
SD Rats, male and Female
20 rats
20 female
20 rats
20 female
20 Rats
20 female
20 rats,
20 female
Control Low Dose Medium Dose High Dose
Study group
Dose level and selection
Admission of test Subs,
oral
Group 1 Group 2 Group 3 Group 4
Findings

22. Non Rodent Animal Species
• Dogs, Pigs and Monkeys are the most commonly used
animals in preclinical studies.
• Ideally , the species of choice should have the same
pharmacokinetic profile as in humans, however, this
information is either incomplete or missing,
• Under such circumstance, select the most sensitive
species for evaluating the safety of the substance.

23. Chronic Toxicity Study
• Species: 2 ( a rodent and a non-rodent).
• In rodents chronic studies are usually for 6 months to 2
years.
• In non-rodents chronic studies are usually for 1 year but
may be longer.
• The length of exposure is somewhat dependent on the
intended period of use in humans.

24. General Toxicology
According to the OECD Guidelines
1. Test Substance
2. Experimental Animals and maintenance
Animal selection,
Species and strain
selection,
Age and Weight,
Husbandry
4. Parameters measured
During the period of test
5. End of Study
6. Data Analysis
7. Data Reporting
Dose level,
Dose selection,
Administration of
test substances
3. Treatment Design
Clinical observation,
Body weight, food intake,
and water intake
Clinical Pathology,
• Hematology,
•Clinical Biochemistry
•Gross necropsyThe evaluation of results will
be with respects to the following:
Clinical observation, Body
weight, food intake, water inyake,
Hematology, Clinical Chemistry,
Gross necropsy, Histopathology
Statistically

25. Clinical Chemistry
• Hepatocellular leakage enzymes:
– ALT, AST, SDH, LDH
• Cholestatic enzymes:
– AP, GGT
• Liver function tests:
– Total bilirubin, direct bilirubin, indirect bilirubin, bile acids,
ammonia
• Pancreatic parameters
– Amylase, lipase
• Lipid parameters
– Cholesterol, triglycerides
• Muscle parameters
– Creatine kinase (CK), AST, ALT, LDH

26. Clinical Chemistry
• Calcium, potassium: Often influenced by kidney
function, kidney parameters should be evaluated
concurrently.
• Kidney Parameters: Urea nitrogen, Creatinine
– Urine specific gravity should be evaluated
concurrently when evaluating renal function.
– Kidney function affect proteins, minerals,
electrolytes, acid-base balance and hematopoietic
parameters.

27. Hematology
• RBC,
• WBC,
• PLT,
• HB,
• Neutrophyl %
• Neutrophyl no
• MCHC,
• HCT
• MCHC
• MCV,

28. Parameters:
Mortality, sign of toxicity, Clinical pathology,
Histology, Weight change, Clinical observation
Goal: To estimate safety margin
Points to consider: Dosing regimen, Duration of clinical trials (Phase I, II,
III), Toxicokinetics, Immunotoxicity
Sub Acute ToxicitySub Acute Toxicity
(OECD Guidelines 407)(OECD Guidelines 407)
Objective: To determine
toxicity after repeated
administration of the test
material
Duration: 28 days daily
dosing, observe daily
Animals Required: 2
species (rodent)

29. Chronic Toxicity (OECD Guideline 452)Chronic Toxicity (OECD Guideline 452)
Objective:
To characterize dose-response
relationships following repeated doses
To identify and characterize specific
organs affected after repeated
administration
Duration:
Rodents - 6 to 24 months;
non-rodents (monkey) - 12 months or
longer or up to 10% of species’
lifespan. Length depends on intended
period of human exposure.
Animals Required: 2 species (rodent
and non-rodent)
Parameters:
Mortality,Clinical pathology, Clinical
observation, Behavioral Assessment,
Histology, Weight change

30. Weight rat

31. Euthanesia

32. Take blood

33. Blood in EDTA
Hematology Analysis

34. Removing organs
Cleaning organs

35. weighing
organs and
collecting data
Histophatology

36. • Data analysis and reporting
• Auditing for compliance
• Data for evaluation for clinical trial

37. You
Thank
For your attention