Acute Toxicity by OECD Guidelines

1. A Seminar on
ACUTE TOXICITY STUDIES AS PER
OECD GUIDLINES
Presented By
Mr. Dalvi Sagar Suresh
First Year M. Pharm (Sem-I)
Department of Pharmacology
Roll No. 02
Pravara Rural College of Pharmacy, Pravaranagar, Loni.
2022-23.

2. Content
• Toxicology
• Animal Toxicity Test
• Acute Oral Toxicity.
• OECD Guidelines For Acute Oral Toxicity.
• Principle Of Limit Test
• Description Of Method
• Procedure
• Observations
• Computation Of Confidence Interval.
• Test Report
• Comparison Of Guidelines.
• Reference

3. Toxicology
• Toxicology is the scientific study of
adverse effects that occur in living
organisms due to chemicals.
• It involves observing and reporting
symptoms, mechanisms, detection and
treatments of toxic substances, in
particular relation to the poisoning of
humans.
• Toxicity tests are mostly used to
examine specific adverse events such
as cancer, cardio toxicity and skin/eye
irritation

4. Animal Toxicity Test
Acute Toxicity 14 Days.
Sub-Acute Toxicity 28 days.
Sub-Chronic Toxicity 3 Months.
Chronic Toxicity 6 Months to 2 Yrs.
Special Toxicity e.g. Carcinogenicity.

5. Acute Oral Toxicity
• Acute toxicity testing is carried out to determine the effect
of single dose on a particular animal species.
• In acute toxicological testing, the investigational product
is administered at different dose levels, and the effect is
observed for 14 days.
• All mortalities caused by the investigational product
during the experimental period are recorded and
morphological, biochemical, pathological and histological
changes in the dead animals are investigated.
• Acute toxicity testing permits the 50 % lethal dose (LD50)
of the investigational product to be determined.

6. Acute Toxicity Studies provide
information on:
• The potential for acute toxicity in humans.
• An estimation of safe acute doses for humans.
• Time course of drug – induced clinical observation.
• The appropriate dosage for multiple – dose toxicity
studies ; and
• Species differences in toxicity

7. OECD Guidelines for Acute Oral
Toxicity
Number Title Date of Adoption
401 Conventional Acute Toxicity
Test
Date of Deletion:
17 Dec, 2002
402 Fixed Dose Procedure 17 Dec, 2001
423 Acute toxic class method 17 Dec, 2001
425 Up and Down Procedure 03 Oct, 2008

8. 425: Acute Oral Toxicity – Up &
Down Procedure (UDP)
• The concept of the up-and-down testing approach was
first described by Dixon and Mood.
• In 1985, Bruce proposed to use an up-and-down (UDP)
for the determination of acute toxicity of chemicals.
• A study comparing the result obtained with the UDP, the
conventional LD50 test and the fixed dose procedure
(FDP, OECD test Guideline 420) was published in 1995.
• The test procedure described in this guideline is of value
in minimizing the number of animals required to estimate
the acute oral toxicity of a chemical.

9. Initial Consideration
• The testing laboratory should consider all available
information on the test substance prior to conducting the study.
• It will include:-
- Identify and chemical structure of the test substance.
- Physical chemical properties;
- Results of any other in vitro or in vivo toxicity tests on the
substance.
• The method permits estimation of an LD50 with a confidence
interval and the results allow a substance to be ranked and
classified according to Globally Harmonised System for the
classification of chemicals which cause acute toxicity.

10. PRINCIPLE OF LIMIT TEST
• The limit test is a sequential test that uses a maximum of
5 animals. A test dose of 2000, or exceptionally 5000
mg/kg. may be used.
• The selection of sequential test plan increases the
statistical power and also has been made to intentionally
bias the procedure towards rejection of the limit test for
compounds with LD50 near the limit dose.

11. PRINCIPLE OF MAIN TEST
• The main test consist of single ordered dose progression in
which animals are dosed, one at a time, at a minimum of 48-
hour intervals.
• The first animal receives a dose a step below the level of the
best estimate of the LD50
• If the animal survives, the dose for the next animal is increased
by a factor of 3.2 times the original dose; it it dies, the dose for
the next animal is decreased by a similar dose progression.
• Each animal should be observed carefully for up to 48 hr
before making a decision on whether and how much to dose
the next animal.
• That decision is based on the 48-hour survival pattern of all the
animals up to that time.
• The LD50 is calculated using the method of maximum
likelihood.

12. Description of Method
Selection Of Animal Species
• The preferred rodent species is the rat, normally female
rats are used.
• When toxicological or toxicokinetic properties of
structurally related chemicals are studied, males are likely
to be more sensitive.
• Criteria for female rats
- They should be nulliparous and non pregnant.
- should be between 8 to 12 years old.
- weight +- 20 % of initial mean weight.

13. Housing and Feeding.
• Temperature - 22 oC ( +- 3 oC).
• Humidity – 50-60 % not exceeding 70 %.
• Lighting should be artificial, the sequence being 12 hr
light and 12 hr dark.
• The animals are housed individually.
• For feeding conventional rodent laboratory diets may be
used with an unlimited supply of drinking water.

14. Preparation of Animals
• The animals are randomly selected, marked to permit
individual identification, and kept in their cages for at least 5
days prior to dosing to allow for acclimatization to laboratory
conditions.
• Ensure that animals are available in the appropriate size and
age range for the entire study.
Preparation of doses
• Test substances should be administered in a constant volume
by varying the concentration of the dosing preparation.
• In rodents, the volume should not normally exceed 1 mi/100g
of body weight however in the case of aqueous solutions, 2
mi/100g body weight can be considered
• For vehicles other than water, the toxicological characteristics
of the ve should be known.

15. PROCEDURE
Administration of Dose
• The test substance is administered in a single dose by
gage using stomach tube or a suitable intubation
cannula.
• Animals should be fasted prior to dosing
• The animals should be weighed and the test substance
administered and the dose is calculated according to
the body weight.
• After the substance has been administered, food may
be withheld for a further 3-4 hrs in rats or 1-2 hr in
mice.

16. Limit test and Main Test
• The limit test is primarily used in situations where the
experimenter has Information indicating that the test
material is likely to be nontoxic, i.e. having toxicity
below regulatory limit doses.
• There is little or no information about its toxicity, or in
which the test material is expected to be toxic, the main
test should be performed.
• Information about the toxicity of the test material can be
gained from knowledge about similar tested compounds
or similar tested mixtures or products, taking into
consideration the Identity and percentage of components.

17. Limit Test
Limit Test at 2000 mg/kg
• Dose one animal at the test dose.
• If the animal dies, conduct the main test to determine the
LD50.
• If the animal survives, dose four additional animals
sequentially so that a total of five animals are tested. If three
animals die, the limit test is terminated and the main test is
performed.
• If an animal unexpectedly dies late in the study, and there are
other survivors. It is appropriate to stop dosing and observe all
animals to see if other animals will also die during a similar
observation period.
• The results are evaluated as follows (O-survival. X-death)

18. Main Test
• Single animals are dosed in sequence usually at 48 h
intervals.
• For selecting the starting dose, information on structurally
related substances and results of any other toxicity tests on
the test material. should be used to approximate the LD50
as well as the slope of the dose- response curve.
• The dose progression factor should remain constant
throughout testing.
• 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).

19. OBSERVATIONS
• Animals are observed individually at least once during the first 30 minutes
after dosing, periodically during the first 24 hours for 14 days.
• The times at which signs of toxicity appear and disappear are recorded.
• Observations should include
- changes in skin and fur.
- eyes and mucous membranes
- respiratory, circulatory
- autonomic and central nervous systems
- behaviour pattern
- salivation
- diarrhoea
- lethargy sleep and coma.
• Animals found in a moribund condition and animals showing severe pain
or enduring signs of severe distress should be humanely killed and time of
death should be recorded.

20. - Bodyweight
• Individual weights are recorded before the test drug
administration and weekly thereafter.
• At the end of the test surviving animals are weighed
and then humanely killed.
- Pathology
• All animals (including those which die during the test
or are removed from the study for animal welfare
reasons) should be subjected to gross necropsy.
• Microscopic examination of organs showing evidence
of gross pathology inanimate surviving 24 or more
hours after the initial dosing may also beconsidered

21. Computation of Confidence Interval
Confidence intervals provides valuable information on the reliability
and utility of the main test that was conducted
• A wide confidence interval indicates that there is more
uncertainty associated with the estimated LD50. The reliability of
the estimated LD50 is low and the usefulness of the estimated
LD50 may be marginal.
• A narrow interval Indicates that there is relatively little
uncertainty associated with the estimated LD50. The reliability of
the estimated LD50 is high and the usefulness of the estimated
LD50 is good.
• This means that if the main test were to be repeated, the new
estimated LD50should be close to the original estimated LD50
and both of these estimates should be close to the true LD50.

22. TEST REPORT
The test report must include the following Information:
• Test substance:
- Physical nature, purity and, where relevant, physical-
chemical properties (Including Isomerization);-
- Identification data, including CAS number.
• Vehicle (if appropriate):-
- Justification for choice of vehicle, if other than water.
• Test animals:
- Species/strain used,
- Microbiological status of the animals, when known;
- Number, age and sex of animals (including where
appropriate, a rationale for use of males Instead of females)
- Source, housing conditions, diet, etc.

23. • Test conditions
- Details of test substance formulation including details of
the physical form of the material levels.
- Details of the administration of the test substance
including dosing volumes and time of dosing administered
- Details of food and water quality (including diet
type/source, water source)..
• Results
- body weight/body weight changes
- Tabulation of response data and dose level for each animal
(i.e, animals showing signs of toxicity including nature,
severity, duration of effects, and mortality)
- Necropsy findings and any histopathological findings for
each animal, if available:-LD50 data.
- |Statistical treatment of results.

24. Comparison of Guidelines
Criteria 420 423 425
Dose 5 – 5000 mg/kg 5 – 5000 mg/kg 1st animal dose
less than
estimated LD50
outcome by factor
3.2
Grouping of
animal
Group of 5 animal
of one sex
Three animals per
group
One animal per
group
Estimation of End
point
1 animal expected
to die
2-3 animal
expected to die
2-3 animals
expected to die
Sighting study Present Absent Absent

25. Reference
1. Dixon W.J. and A.M. Mood. (1948). A Method for Obtaining and
Analyzing Sensitivity Data. J. Amer. Statist. Assoc., 43, 109-126.
2. Dixon WJ. The Up-and-Down Method for Small Samples (1965).
J. Amer. Statist. Assoc. 60, 967-978.
3. Bruce R.D. (1985), An Up-and-Down Procedure for Acute
Toxicity Testing Fundamental Application Toxicity. 5, 151-157.
4. ASTM (1987), E 1163-87, Standard Test Method for Estimating
Acute Oral Toxicity
5. OECD (2000). Guidance Document on the Recognition,
Assessment and Use of Clinical Signs as Humane Endpoints for
Experimental Animals Used in Safety Evaluation. Environmental
Health and Safety Monograph Series on Testing and Assessment
No 19.