General Toxicology

Dr. Noor Rain Abdullah

Herbal Medicine Research Center
Institute for Medical Research,
Kuala Lumpur
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

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

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

1. Test Substance
Animal selection,
Species and strain
2. Experimental Animals and maintenance selection,
Age and Weight,
Dose level, Husbandry
Dose selection,
Administration of 3. Treatment Design
test substances

4. Parameters measured Clinical observation,

During the period of test Body weight, food intake,
and water intake

Clinical Pathology,
5. End of Study • Hematology,
•Clinical Biochemistry
The evaluation of results will •Gross necropsy
be with respects to the following:
Clinical observation, Body 6. Data Analysis
weight, food intake, water inyake, Statistically
Hematology, Clinical Chemistry,
Gross necropsy, Histopathology
7. Data Reporting
 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
 Acute Toxicology
Rats, female

Group 1 Group 2

Control Study group 5 g/kg

5 rats 5 rats
Dose level and selection

Admission of test Subs, orally

Findings

OECD Guidelines 420, 425

 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
 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
 Acute Toxicity (OECD Guideline 420, 425)

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)

Parameters:
Mortality, Clinical pathology, Gross necropsy, Weight change,
Clinical observations
Points to consider:
Dose selection for repeat dose studies
 What is subacute toxicity
• The effect of exposure from 1 day to 30
days
 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.
 Subacute (Guidelines 407)
SD Rats, male and Female
Study group

Group 1 Group 2 Group 3 Group 4

Control Low Dose Medium Dose High Dose

5 rats 5 rats 5 Rats 5 rats,

5 female 5 female 5 female 5 female

Dose level and selection

Admission of test Subs,

oral

Findings
 What is chronic toxicity
• Daily exposure for 3 months or more
 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
 Chronic (Guidelines 452)
SD Rats, male and Female
Study group

Group 1 Group 2 Group 3 Group 4

Control Low Dose Medium Dose High Dose

20 rats 20 rats 20 Rats 20 rats,

20 female 20 female 20 female 20 female

Dose level and selection

Admission of test Subs,

oral

Findings
 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.
 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.
 General Toxicology
According to the OECD Guidelines

1. Test Substance
Animal selection,
Species and strain
2. Experimental Animals and maintenance selection,
Age and Weight,
Dose level, Husbandry
Dose selection,
Administration of 3. Treatment Design
test substances

4. Parameters measured Clinical observation,

During the period of test Body weight, food intake,
and water intake

Clinical Pathology,
5. End of Study • Hematology,
•Clinical Biochemistry
The evaluation of results will •Gross necropsy
be with respects to the following:
Clinical observation, Body 6. Data Analysis
weight, food intake, water inyake, Statistically
Hematology, Clinical Chemistry,
Gross necropsy, Histopathology
7. Data Reporting
 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
 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.
 Hematology
• RBC,
• WBC,
• PLT,
• HB,
• Neutrophyl %
• Neutrophyl no
• MCHC,
• HCT
• MCHC
• MCV,
Sub Acute Toxicity
(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)

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
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
Weight rat
Euthanesia
Take blood
 Hematology Analysis

Blood in EDTA
Removing organs

Cleaning organs
weighing
organs and
collecting data

Histophatology
• Data analysis and reporting
• Auditing for compliance
• Data for evaluation for clinical trial
Thank
You

For your attention