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Expt. 10 Determination of acute skin irritation corrosion of a test substance

The document outlines experiment no. 10 for determining acute skin irritation and corrosion of a test substance using guinea pigs, which are the chosen animals for skin sensitization tests. It details the objective, procedure involving an induction and challenge phase, and grading scale for evaluating sensitization potential based on skin reactions. Additionally, it discusses the use of Freund's Complete Adjuvant as an immunopotentiator and its regulatory considerations due to toxicity concerns.

Education◦
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 1
Overview of Discussion • Objective • Principle • Requirements • Procedure • Observation table • Result and interpretation • Additionalinformation 2
Objective To perform acute toxicity testing for topical preparations. 3
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. 4
Requirements  Animals: Guinea pigs, both sexes.  Chemicals: Freund's complete adjuvant (FCA).  Miscellaneous: Physiological saline, syringe, shaving kit, Whatman filter paper no. 1 5
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. 6
(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
 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. 8
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
Fig. Experimental design for dermal sensitization testing: guinea pig dorsal area 10
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Observation table 13 Group Scoring at induction phase Scoring at challenging phase At 24 hours At 48 hours At 24 hours At 48 hours Control Test
Result and interpretation Comparison of scoring between study groups estimates the sensitization potential of test drug. 14
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. 15
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
ANY QUESTION??? 17