OECD GUIDELINES

Presented by P.Balaji (Research Scholar) Department of Pharmacy Annamalai University

OECD
ORGANISATION FOR ECONOMIC CO-OPERATION AND DEVELOPMENT
1. Toxicity tests - investigation of a new drug involve Acute, Sub acute and chronic toxicity. 2. Acute toxicity is involved in estimation of LD50 (the dose which has proved to be lethal (causing death) to 50% of the tested group of animals).

OECD 420 , 423 & 425

OECD General Guidelines

Traditional methods for assessing acute toxicity use death of animals as an endpoint. In 1984, a new approach - acute toxicity testing was suggested by the British Toxicology Society based on the administration at a series of fixed dose levels.

Avoid using death of animals & Observation sign of toxicity

General Guidelines cont

INITIAL CONSIDERATIONS: Only moderately toxic doses are used Administration of doses - lethal should be avoided o cause marked pain and distress

o due to corrosive or severely irritant actions

o showing signs of severe and enduring distress

Hazardous - Globally Harmonised System (GHS) for the classification of chemicals which cause acute toxicity

General Guidelines cont

Testing laboratory: All available information on the test substance. o Identity and chemical structure of the substance o Physico-chemical properties o The results of any other in vitro or in vivo toxicity tests on the substance

o Toxicological data on structurally related substances

o Above information is necessary human & starting dose

General Guidelines cont

Selection of animal species: Rodent species Females are used (More Sensitive)

Test is conducted in males, adequate justification should be provided.

Nulliparous and non-pregnant 8 and 12 weeks old

General Guidelines cont

Housing and feeding conditions: Room Temp: 22C (+3C) RH is at least 30% and preferably not exceed 70%

Lighting should be artificial 12h (D/L)

Feeding conventional laboratory diets & Water

Group-caged by dose, but the number of animals per cage & Clear observations of each animal

General Guidelines cont

Preparation of doses: Rodents, the volume should not normally exceed 1mL/100g of body weight Use of an aqueous Solution / suspension / recommended emulsion is

For vehicles - toxicological characteristics of the vehicle should be known

General Guidelines cont

Administration of doses: Gavage using a stomach tube or a suitable intubation canula

Unusual circumstance that a single dose is not possible - dose may be given in smaller fractions over a period not exceeding 24 hours Animals should be fasted prior to dosing

After the substance has been administered, food may be withheld

General Guidelines cont

OBSERVATIONS: After dosing at least once during the first 30 minutes,

Special attention given during the first 4 hours,

periodically during the first 24 hours / 14 days

Signs of toxicity appear and disappear are important

General Guidelines cont

OBSERVATIONS: (Cont) Skin and fur Eyes and mucous membranes Respiratory & circulatory Autonomic and central nervous systems Somatomotor activity and behaviour pattern

Tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma

Urine analysis

General Guidelines cont

Pathology: All test animals (survive / death) gross necroscopy (Microscopical examinations of each organs evidence of gross pathology)

Organs - brain, colon, heart, kidneys, liver, lungs, oesophagus, rectum, sciatic nerve, spleen, sternum with bone marrow, stomach, thyroid / parathyroid, trachea, urinary bladder and uterus. They were subjected to histopathological examination

General Guidelines cont

Body weight: Individual weights of animals before test substance admn, one week after & end of the test survive animals

Wt. of the organs - liver, kidneys, adrenals, epididymis, thymus, spleen, brain, heart, uterus and testes/ovaries

OECD 420
The original Guideline 420 was adopted in July 1992 Fixed dose method does not require the death of animals as an endpoint Animals used: 5 female rats Dose: 5, 50, 300 & 2000 mg / kg Upper fixed dose level of 5000 mg/kg - reasons of animal welfare concern, testing of animals in GHS Category 5 ranges Sighting study & main study carried out

OECD 420 cont

5mg/kg

1 animal
Animal dies Terminate the study Further confirm GHS category 1

Sighting study

Second animal dosed at 5 mg/kg

OECD 420 cont

Main study

5 animals

One animal from the sighting study dosed at the selected dose level together with an additional four animals

OECD 420 cont

OECD 420 cont

OECD 420 cont

OECD 420 cont

OECD 423 (Acute toxic class method)

Guideline 423 was adopted in March 1996

Second alternative to the conventional acute toxicity test

The expected number of deaths is typically not more than 3 Similar to OECD 420 No sighting study Mortality is endpoint

OECD 423 cont

Animals Used: 6 Female Animals

Dose: 5 , 50 , 300 & 2000 mg / kg (Starting at any dose)

PRINCIPLE OF THE TEST: o no further testing is needed, o dosing of three additional animals, with the same dose o dosing of three additional animals at the next higher or the next lower dose level.

OECD 423 cont

OECD 423 cont

OECD 423 cont

OECD 423 cont

OECD 423 cont

TESTING AT DOSES ABOVE 2000 MG/KG:
5000 mg / kg

3 Animals

Ist animal dies

Dosing

2000 mg / kg

Ist animal survives

Dosing

5000 mg / kg

5000 mg / kg

LD 50 value

Only One of the three animal dies

OECD 425
1985 Bruce Acute toxicity of chemicals Up & down method Minimizing the number of animals

Revision of guidelines 420 & 423

Start dose: Believed just below LD50 or 175 mg/kg

Increase-decrease usually x 3.2 (dose progression of factor) antilog of 1/(the estimated slope of the dose-response curve), 48 h between animals

OECD 425 cont

Find dose level - outcome - death or no death LD50 is calculated 5 Female animals used

Test dose of 2000 or exceptionally 5000 mg/kg, may be used

Limit test & main test should be performed

OECD 425 cont

Limit test at 2000 MG/KG:
Test dose (1 + 4)

1 Animal

Animal dies

Conduct

Main test

Animal survives

LD 50

4 Animals

3 Animal dies

Limit test is terminated & main test performed

OECD 425 cont

The LD50 is less than the test dose (2000 mg/kg or 5000 mg / kg) when three or more animals die. o xoxx , o xxox , o oxxx Main test

The LD50 is greater than the test dose (2000 mg/kg or 5000 mg / kg) when three or more animals survive. o oooo , o oxoo , o oxoo Limit test

5000 mg / kg

OECD 425 cont

First animal

Test dose
Survive Receives

Second animal

Receives

Higher dose

Dies
Second animal Lower dose

The dose progression factor 3.2 and should remain constant throughout testing.

OECD 425 cont

When there is no information on the slope of the substance to be tested, a dose progression factor of 3.2 is used. Using the default progression factor, doses would be selected from the sequence 1.75, 5.5, 17.5, 55, 175, 550, 2000 (or 1.75, 5.5, 17.5, 55, 175, 550, 1750, 5000 for specific regulatory needs). If no estimate of the substances lethality is available, dosing should be initiated at 175 mg/kg.

REFERENCES
www.oecd-ilibrary.org/environment/test-no420-acute-oral-toxicity-fixed-doseprocedure_9789264070943-en www.oecd.org/dataoecd/17/50/1948370.pdf www.oecd.org/dataoecd/17/51/1948378.pdf

Thank You