Acute dermal toxicity-402

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 ± 3 o C for rats & 20 ± 3 o C 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

10. 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.

11. REFERENCES RCC_TOX_006_V03. OECD Guidelines for the Testing of Chemicals, Number 402 "Acute Dermal Toxicity", adopted 24 th 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