This presentation will help to understand the principle and purpose of acute dermal toxicity study in animal model.

1. ACUTE DERMAL TOXICITY
OECD/OCDE : 402 Adopted: 9 October 2017
Dr. Ajay Mandal

2. PRINCIPLE & PURPOSE
• The original acute Dermal Toxicity Guideline TG 402 was adopted in 1987.
• A number of recent publications have analyzed the results of acute oral and
dermal toxicity studies for hundreds of pesticide active substances and
thousands of industrial chemicals, finding that regulatory classifications
based on oral acute toxicity data were equivalent to or more severe than those
derived from dermal data in more than 98 percent of cases, calling into
question the value of routine testing for this endpoint.
• Based on the recommendations of several expert meetings, international
agreement had been reached on harmonised LD50 cut-off values for the
classification of chemicals.

3. A REVISION OF OECD 402 WAS CONSIDERED TIMELY BECAUSE:-
 i) Testing in one sex (usually females) is generally considered sufficient.
 ii) In order for a point estimate to be meaningful, there is a need to estimate
confidence intervals.
 The stepwise procedure in OECD Test Guideline 402, with the use of up to 3
animals of a single sex per step, has been adapted from the acute toxic class
method and the fixed dose procedure set out in OECD Test Guideline 420.
 Depending on the mortality and/or the moribund status of the animals, further
steps may be necessary to allow judgement on the acute toxicity of the test
chemical.
 The test chemical is administered to single animals in a sequential manner with
two animals used at any selected dose level in the main study.

4. TEST SYSTEM
 Selection of Species – Rat, Other Species – Justification Required
 Animal Procurement – Healthy young adult animals should be procured from CPCSEA
approved Animal Breeding Facility
 Body Weight – Rat body weight (200g-300g), Guinea pig body weight (350g-450g), Rabbit
body weight (1.5kg-3.0kg)
 Age – Rat (8-12 weeks old)
Guinea pig (5-6 weeks old)
Rabbit (at least 12 weeks old)
 Sex - Male / Female (Nulliparous & Non-Pregnant)
 Acclimatization – Minimum 5 days
 Randomization - Manually
 Housing - Caged individually
 Identification Temporary – Animal No. : Tip of Tail with Red Marker pen
Permanent – Animal No.: Base of tail with Black Marker pen
 Note: Cage should be identified by – Study No, Test item code, Study code, cage No, species, sex,
No. of animals, Dose/ Conc, Group No. & Animal No.

5. HUSBANDRY
Temperature – 22 ± 3oC for rats & 20 ± 3oC for rabbits and Guinea Pigs.
Relative Humidity – 30 to 70%
Air Changes – At least 10-15 ACH
Photo Period – 12 hours dark & 12 hours light
Bedding – Sterilized Paddy Husk
Feed – Ad libitum
Water – Ad libitum
PREPARATION OF TEST ITEM
Solids – Pulverized (if necessary) before used and moistened with distilled water or
vehicle.
Liquid – applied directly with out dilution.

6. PREPARATION OF TEST ANIMALS
• One day before application of test item, 10 % of the total body surface area of the animals
will be clipped with electric clipper/shaved with razor blade/ depilated with VEET cream.
•The position of clipping will be the area starting at the scapulae (shoulders) to the wing of
the Ilium (hipbone) and half way down the flank on each side.

7. TEST ITEM APPLICATION
• Test item should be applied uniformly over a 10 % area of total body surface (approx
4cm x 4cm area of skin covered by test item) with porous gauge dressing and non
irritating tape for a period of 24 hours.
• After 24 hours exposure period, residual test item should be removed with distilled
water.
BODY WEIGHTS
•Body weights should be taken –
Animal procurement
Randomization
Day 0 (prior to Administration), Day 7, Day 14 and at death

8. DOSE RANGE FINDING STUDY
 When there is no or insufficient information on a test chemical, a dose-range
finding study using 1 animal at a starting dose of 200 mg/kg body weight is
recommended to minimise animal use and optimise the study design
 Based on the outcome in the range-finding study, the main study can be
conducted with 2 further animals to confirm the classification outcome.
LIMIT TEST
 If information is available for the test chemical, a different starting dose may be
chosen, e.g. 50, 1000 or 2000 (akin to a limit dose) mg/kg bw, following the
same procedure (range-finding study followed by main study), based on the
GHS Categories for acute dermal toxicity.

9. FLOWCHART FOR THE TESTING PROCEDURE

11. OBSERVATIONS
 Animals are observed immediately after dosing at least once during the first 30 minutes,
periodically during the first 24 hours, with special attention given during the first 2 to 6 hours
after the beginning of the exposure period and daily thereafter, for a total of 14 days.
 Animals found in a moribund condition and animals showing severe pain and/or enduring
signs of severe distress should be humanely killed without delay. When animals are killed for
humane reasons or found dead, the time of death should be recorded as precisely as possible.
OBSERVATIONS SHOULD INCLUDE:-
 Changes in skin, fur, eyes, mucous membranes, also respiratory, circulatory, autonomic,
central nervous systems, somatomotor activity and behaviour pattern.
 Attention should be directed to observations of tremors, convulsions, salivation, diarrhoea,
lethargy, sleep and coma.
 In addition, the treatment site may be observed at 24, 48 and 72 hours after removal of test
chemical using the Draize criteria, as these data may be useful for waiving the need for a
separate in vivo skin irritation study.

12. REFERENCES
 RCC_TOX_006_V03.
 OECD Guidelines for the Testing of Chemicals, Number 402 "Acute Dermal
Toxicity", adopted 24th February 1987 .
 CPCSEA Guidelines for Laboratory Animal Facility. Indian Journal of Pharmacology.
2003; 35: 257-274.
 Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal
Resources. Commission on Life Sciences. National Research Council. National
Academy Press. Washington, D.C. 1996.
 Guidance Document on the Recognition, Assessment and Use of Clinical signs as
humane endpoints for Experimental Animals used in Safety Evaluation.
ENV/JM/MONO(2000)7. OECD, December, 2000.
 EC Guideline - B.3. Acute Toxicity (Dermal).
 EPA - Health Effects Test Guidelines OPPTS 870.1200 Acute Dermal Toxicity.
 WHO TOXICITY CLASSIFICATION