The method is found in the main body of the document.
It is also known as the Magnusson-Kligman Test
If the test article is to be tested directly, the preparation is to be according to an associated annex. A note has been inserted discussing a specific method for extracting polymers.
Healthy albino guinea pigs of either sex from a single strain between 300 and 500 grams should be used. Females shall be nulliparous and not pregnant.
The number of animals used should be a minimum of 10 test animals for a powder, liquid or each extraction vehicle. A minimum of 5 control animals for a powder, liquid or for each extraction vehicle. Additional animals may be required for preliminary tests.
The fur should be clipped prior to treatment.
Dosages
0.1 ml per site for intradermal injections.
Saturated filter paper or gauze under an occlusive dressing for topical applications.
Preliminary tests:
Main test:
Three Phases
Intradermal Induction Phase:
Topical Induction Phase:
If there was no irritation observed in the topical preliminary test, apply 10% sodium dodecyl sulfate (SDS) in petrolatum 24 (+/-2) hours before topical application of the test article or extract (see below). Perform the same procedure to the control animals. Freshly prepared extracts should be used. If the extracts are stored for greater than 24 hours the stability needs to be verified. Apply the highest concentration that caused slight erythema but didn’t affect the animals in the preliminary topical test to filter paper over the shoulder blades topically and secure with occlusive dressing 7 (+/-1) days after the intradermal induction phase. The dose should be removed after 48 hours. Control animals receive the the vehicle or carrier.
Challenge Phase:
Apply the highest concentration that caused no erythema in the topical preliminary test to the flanks of both the test and the control animals 14 (+/- 1) days after the completion of the topical induction phase. The dose should be applied for 24 (+/- 2) hours via patches as in the topical induction phase under an occlusive dressing.
Observe the dosed sites of all animals 24 (+/- 2) and 48 (+/- 2) hours after the removal of the patches. Grading is on a 5 point scale ranging from no(0), very slight(1), well-defined(2), moderate(3), to severe(4) for both erythema and edema. Grades in test animals greater than the grades observed in the control animals indicate sensitization. Questionable results may warrant a rechallenge 1 to 2 weeks later following the same procedures although a new FCA treated control group would be necessary.
It is also known as the Magnusson-Kligman Test
If the test article is to be tested directly, the preparation is to be according to an associated annex. A note has been inserted discussing a specific method for extracting polymers.
Healthy albino guinea pigs of either sex from a single strain between 300 and 500 grams should be used. Females shall be nulliparous and not pregnant.
The number of animals used should be a minimum of 10 test animals for a powder, liquid or each extraction vehicle. A minimum of 5 control animals for a powder, liquid or for each extraction vehicle. Additional animals may be required for preliminary tests.
The fur should be clipped prior to treatment.
Dosages
0.1 ml per site for intradermal injections.
Saturated filter paper or gauze under an occlusive dressing for topical applications.
Preliminary tests:
- Not required for undiluted extracts using typical solvents.
- Used for the purpose of establishing concentrations in main test.
- Pretreat the animals with Freund’s Complete Adjuvant (FCA).
- 3 topical preliminary animals receive a range of dose concentrations.
Main test:
Three Phases
Intradermal Induction Phase:
- 3 pairs of 0.1 ml injection.
- First pair is a 1 to 1 mixture of FCA and the vehicle or carrier.
- The second pair is the concentration of the test article determined to not cause extensive skin destruction in the intradermal preliminary test or the extraction vehicle alone in the case of the control animals.
- The third pair is a 1 to 1 mixture of what was injected in the second pair with FCA.
Topical Induction Phase:
If there was no irritation observed in the topical preliminary test, apply 10% sodium dodecyl sulfate (SDS) in petrolatum 24 (+/-2) hours before topical application of the test article or extract (see below). Perform the same procedure to the control animals. Freshly prepared extracts should be used. If the extracts are stored for greater than 24 hours the stability needs to be verified. Apply the highest concentration that caused slight erythema but didn’t affect the animals in the preliminary topical test to filter paper over the shoulder blades topically and secure with occlusive dressing 7 (+/-1) days after the intradermal induction phase. The dose should be removed after 48 hours. Control animals receive the the vehicle or carrier.
Challenge Phase:
Apply the highest concentration that caused no erythema in the topical preliminary test to the flanks of both the test and the control animals 14 (+/- 1) days after the completion of the topical induction phase. The dose should be applied for 24 (+/- 2) hours via patches as in the topical induction phase under an occlusive dressing.
Observe the dosed sites of all animals 24 (+/- 2) and 48 (+/- 2) hours after the removal of the patches. Grading is on a 5 point scale ranging from no(0), very slight(1), well-defined(2), moderate(3), to severe(4) for both erythema and edema. Grades in test animals greater than the grades observed in the control animals indicate sensitization. Questionable results may warrant a rechallenge 1 to 2 weeks later following the same procedures although a new FCA treated control group would be necessary.
