A detailed methodology, principle , observations and TEST RESULTS

1. ACUTE DERMAL TOXICITY
STUDIES
AS PER OECD GUIDELINES
PREPARED BY : JESHICA P. BULSARA
M.PHARM SEM II
DEPARTMENT OF PHARMACOLOGY
SSR COLLEGE OF
PHARMACY,SAYLI,SILVASSA

2. INTRODUCTION
• The original acute Dermal Toxicity Guideline TG 402 was adopted in 1987.
402 : ACUTE DERMAL TOXICITY - FIXED DOSE PROCEDURE
• A revision of TG 402 was considered timely because:
i) Testing in one sex (usually females) is generally considered sufficient, and
ii) In order for a point estimate to be meaningful, there is a need to estimate confidence
intervals (CI).
• 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.
• The test chemical is administered to single animal in a sequential manner with two animals
used at any selected dose level in the main study.
• The procedure is reproducible, uses very few animals and is able to rank test chemicals in a
similar manner to the other acute toxicity testing methods.

3. INITIAL CONSIDERATIONS
• The testing laboratory should consider all available information on the test substance
prior to conducting the study.
• It will include
- Identity and chemical structure of the test substance;
- Physical chemical properties;
- Results of any other in vitro or in vivo toxicity tests on the substance;
- Toxicological data on structurally related substances or similar mixtures;
- Anticipated use(s) of the substance.
• Test chemicals should not be administered at doses that are known to cause marked
pain and distress due to potential corrosive or severely irritant actions.
• Moribund animals or animals obviously in pain or showing signs of severe and
enduring distress shall be humanely killed, and are considered in the interpretation of
the test results in the same way as animals that died on test.

4. PRINCIPLE
• Groups of animals, of a single sex, are exposed to the test chemical in a stepwise
procedure using the appropriate fixed doses.
• Groups of animals may be tested at higher or lower fixed doses, depending on the
presence or absence of signs of toxicity or mortality.
• This procedure continues until the dose causing toxicity or no more than one death is
identified, or when no effects are seen at the highest dose or when deaths occur at the
lowest dose.

5. TEST SYSTEM
• Selection of species – Rats , Other species- justification required.
• Animal procurement – Healthy Adult animals should be procured from CPCSEA
approved animal Breeding facility
• Body weight – Rat (200-300g) , Guinea pig (350-450g) , Rabbit(1.5-3kg)
• Age – Rats (8 to12 weeks)
Guinea pigs (5 to 6 weeks old)
Rabbits (at least 12 weeks old)
• Sex – Females ( nulliparous and non-pregnant)
Males (adequate justification required) but can be used

6. • Acclimatization – Minimum 5 days
• Randomization – Manually
• Housing – Caged individually
HUSBANDARY
• Temperature – 22 ± 3°C for rats , 20 ± 3°C for guinea pigs and rabbits.
• Relative humidity – 30 to 70%
• Photoperiod – 12hrs dark 12hrs light
• Feed – laboratory diets with an unlimited supply of drinking water.
PREPARATION OF TEST SYSTEM
Solids – Pulverised before used and moistened with distilled water or vehicle.
Liquids – Applied directly

7. PREPARATION OF ANIMALS
• The animals are acclimatized to the laboratory conditions for at least five
days prior to the start of the study.
• On the day before administration of the test chemical, all fur should be
removed from the dorsal/flank area of the test animals by closely clipping.
• Care must be taken to avoid abrading the skin, which could alter its
permeability.

8. TEST ITEM APPLICATION
• Test chemicals should be held in contact with the skin with a porous gauze
dressing and nonirritating tape throughout a 24-hour exposure period.
• The test site should be further covered in a suitable manner to retain the
gauze dressing and test chemical and ensure that the animals cannot ingest
the test chemical.
• During the 24-hour exposure period animals may be caged individually in
order to avoid oral ingestion of the test chemical by other animals in the
cage.
• At the end of the exposure period, residual test chemical should be
removed, where practicable using water or an appropriate solvent.

9. • When there is no or insufficient information on a test chemical, a dose-range
finding study using ONE animal at a starting dose of 200 mg/kg body
weight is recommended to minimize animal use and optimize 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.
• 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
DOSE RANGE FINDING STUDY

10. 1 animal
50mg/kg
1 animal
2000mg/kg
1 animal
1000mg/kg
1 animal
200mg/kg
A B BABABA
GHS Cat 1 200 200 1000 2000
Main
study
starting
dose
START
RANGE FINDING STUDY A
B
Death
Toxicity or no death

12. GHS CRITERIA FOR ACUTE DERMAL TOXICITY
• Category 1
LD50 ≤ 50mg/kg
• Category 2
LD50 > 50mg/kg ≤ 200mg/kg
DANGER
Fatal in contact with skin
• Category 3
LD50 > 200mg/kg ≤ 1000mg/kg
DANGER
Toxic in contact with skin
• Category 4
LD50 > 1000mg/kg ≤ 2000mg/kg
WARNING
Harmful in contact with the skin
• Category 5
LD50 > 2000mg/kg ≤ 5000mg/kg
WARNING
Harmful in contact with the skin

13. 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.

14. Observations should include :
• changes in skin and fur,
• eyes and mucous membranes,
• respiratory, circulatory,
• autonomic and central nervous systems,
• somatomotor activity
• behaviour pattern
• 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.

15. PARAMETERS 0 1 2 3
Skin Erythema and Redness , swelling,
discharge ,
, hemorrhage
Fur Shining Matte Ruffled
Eyes Clear and
clean
Unclear and sticky Closed / semi closed
Posture Normal Hunched Massively Hunched
Mucous
membrane
Normal Lesions
Secretions &
Excretion
No Yes
Lacrimation No Yes

16. PARAMETERS 0 1 2 3
Pupil size Normal Abnormal
Unusual
respiration
Breathed
normally
Slightly changed Accelerated
breathing
Strongly
accelerated ,
or irregular
Change in gait Normal Slight In coordinated
OR reluctance
to move
Staggering
OR limb
dragging
Response while
handling
The rat’s
muscles are
relaxed
Struggling Squeaking Biting during
handling
Stereotype Excessive
grooming
Repetitive circling
Tonic clonic
convulsions
No Yes

17. Test Report
• Test chemical - source, lot number, limit date for use, if available; - stability
of the test chemical itself, if known;
- solubility and stability of the test chemical in vehicle, if known;
Mono-constituent substance:
- Physical appearance and additional relevant physicochemical properties (e.g.
acid/alkaline reserve);
- Chemical identification, such as IUPAC or CAS name, CAS number, SMILES or InChI
code, structural formula,
- Purity,
- Chemical identity of impurities as appropriate and practically feasible, etc.
Multi-constituent substance, UVCBs and mixtures:
- characterized as far as possible by chemical identity (see above), quantitative
occurrence and relevant physicochemical properties of the constituents and mixture.

18. Vehicle (if appropriate):
justification for use of vehicle and 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, historical data etc.;
- Details of food and water quality (including diet type/source, water source);
method of randomization in animal selection.
Test conditions:
- Details of test chemical formulation, including details of the physical form of the test
chemical administered;
- Details of the administration of the test chemical and the treatment site including dosing
volumes, area of application, and duration of exposure;
- The rationale for the selection of the starting dose;

19. Parameters OECD EPA SCHEDULE Y
No of animals 3 5 Not mentioned
Species Rats . Others
( justification
required)
Rats , Rabbit ,
Guinea pigs
Rats , rabbits
COMPARISON WITH OTHER GUIDELINES

20. 1. OECD Guidelines for the Testing of Chemicals, Number 402 "Acute Dermal
Toxicity", adopted 9th October 2017.
2. CPCSEA Guidelines for Laboratory Animal Facility. Indian Journal of
Pharmacology. 2003; 35: 257-274.
3. EPA - Health Effects Test Guidelines OPPTS 870.1200 Acute Dermal Toxicity.
4. “Schedule Y” – CDSCO.
REFERENCES