Objective Principle Requirements Procedure Observation table Result and interpretation Additional information

1. Experiment No. 10
Determination of acute skin irritation
corrosion of a test substance
Mr. Vishal Balakrushna Jadhav
Assistant Professor (Pharmacology)
GES’s Sir Dr. M. S. Gosavi COPER, Nashik-5
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2. Overview of Discussion
• Objective
• Principle
• Requirements
• Procedure
• Observation table
• Result and interpretation
• Additionalinformation
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3. Objective
To perform acute toxicity testing for topical preparations.
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4. Principle
Guinea pigs are the animals of choice for skin sensitization tests.
Guinea Pig Maximization Test (GPMT) is among the mostly
used methods for testing of chemicals topically.
The skin sensitization procedure is an immunological process in
which the test animal acquires a specific allergic sensitivity to
test substance on repeated exposure and the response is
manifested as increased erythema (abnormal redness of the
skin resulting from dilation of blood vessels). At first test
animals are injected the test substances by intradermal or
epidermal route (induction exposure). The animals are then
exposed to a test dose. The extent and degree of skin
reaction in test animals is compared with control animals
which undergo sham treatment during the period.
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5. Requirements
 Animals: Guinea pigs, both sexes.
 Chemicals: Freund's complete adjuvant (FCA).
 Miscellaneous: Physiological saline, syringe, shaving kit,
Whatman filter paper no. 1
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6. Procedure
 Weigh and select healthy young adult animals of both sexes for the
study. Female animals should be nulliparous (having never given birth)
and non-pregnant. Take 10 animals for test group and 5 animals for
controlgroup. Animals should be weighed before and after the test.
 Procedure involves testing the drug in two phases, i.e. induction phase
and challenge phase.
A)Induction phase
For test group
 Prepare three pairs of intradermal injections. Injection 1 contains 1:1
(v/v) mixture of Freund's complete adjuvant (FCA)/water or
physiological saline. Injection 2 contains a test substance mixed
inappropriate vehicle at selected concentration and injection 3 contains
test substance at required concentration formulated in 1:1 (v/v) mixture
of FCA/water or physiological salt solution.
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7. (For injection 3 if test substance is water soluble, dissolve the same in
aqueous phase first then mix with FCA. If test compound is lipid soluble
then suspend the drug in FCA and then in aqueous phase).
 Shave the shoulder portion and caudal region of the animal either by
clipping, shaving or chemical depilation. Inject the first and second
injection through intradermal route at a dose of 0.1 ml on both the
sides of shoulder (nearest the head) and third injection on the caudal
area.
For control group
 Inject three pairs of 0.1 ml intradermal injections similarly at the same
sites as in treatment group without test drug in preparations.
 Observe the treated sites of each animal and score at 24 and 48 hours
afterinjection.
 After 7 days of primary induction phase, the test area is once again
shaved cleanly and the test drug is administered topically by applying
test drug saturated 2 cm x4 cm section Whatman filter paper no. 1 over
the sites treated previously for 48 hours. 7

8.  The animals in the control group are treated in a similar manner but
without test drug. After 48 hours the treated sites are examined and
scored.
B) Challenge phase
After 14 days of topical induction, the flanks of animals in both the
groups are shaved. A patch of 2 cm× 2 cm section of Whatman filter
paper no. 1 loaded with highest non-irritating concentration of the
test drug is placed on the left flank and only vehicle loaded paper is
to be placed on right flank (in case of solids they should be
grounded finely and mixed in vehicle; in case of liquid it should be
used in undiluted form). These patches are held in contact with
flanks by occlusive dressing for 24 hours. After 24 and 48 hours the
patch will be removed, examined and scored.
Sensitization potential is determined by positive response showed by
percentage of animals involved in the study. A score 1 or greater for
redness is considered as a positive reaction of test substance.
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9. 9
Table- Magnusson and Kligman grading scale for the
evaluation of challenge patch test reactions
Score Evaluation
0 No visible change
1 Discrete or patchy erythema
2 Moderate and confluent erythema
3 Intense erythemaand swelling

10. Fig. Experimental design for dermal sensitization
testing: guinea pig dorsal area
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13. Observation table
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Group
Scoring at
induction phase
Scoring at challenging
phase
At 24 hours At 48 hours At 24 hours At 48 hours
Control
Test

14. Result and interpretation
Comparison of scoring between study groups estimates the
sensitization potential of test drug.
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15. Additionalinformation
Freund's adjuvant
Freund's adjuvant is a solution of antigen emulsified in mineral
oil and used as an immunopotentiator (booster). The complete
form, Freund's Complete Adjuvant (FCA or CFA) is composed
of inactivated and dried mycobacteria (usually M. tuberculosis).
The incomplete form (FIA or IFA) lacks the mycobacterial
components (hence just the water in oil emulsion). It is named
after Jules T. Freund.
Regulation
Freund's complete adjuvant is effective in stimulating cell-
mediated immunity and leads to potentiation of T helper cells
that leads to the production of certain immunoglobulins and
effector T cells. Its use in humans is forbidden by regulatory
authorities, due to its toxicity. Even for animal research there
are currently guidelines associated with its use, due to
its painful reaction and potential for tissue damage.
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16. Injections of FCA should be subcutaneous or intraperitoneal,
because intradermal injections may cause skin
ulceration and necrosis; intramuscular injections may lead to
temporary or permanent muscle lesion, and intravenous
injections may produce pulmonary lipid embolism.
Effects
When administered to diabetes prone non-obese diabetic (NOD)
mice, FCA prevented juvenile-onset diabetes. When combined
with spleen cells, FCA was said to have reversed diabetes. In
2006, these claims were confirmed that even without spleen
cells FCA can restore insulin producing beta cells in pancreas of
NOD mice.
It has also been investigated in an animal model of Parkinson's
disease, or used in emulsion with myelin oligodendrocyte
glycoprotein (MOG), a peptide inducing experimental
autoimmune encephalomyelitis (EAE) in animal studies for
efficacy testing of multiple sclerosis treatments. 16

17. ANY QUESTION???
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