Sensitization Test
Sensitization test：Selection and performance of tests that can evaluate skin sensitization reactions associated with the safety of medical materials and devices. There are currently three animal tests for the determination of potential skin sensitization to chemicals, including two guinea pig tests and one mouse test. To date, the two methods most commonly used to examine skin sensitization are the guinea pig maximum dose test (GPMT) and the closed patch test (Beuhler). The GPMT is the most sensitive method. The closed patch test is suitable for topical application of the product.
Maximum dose testing
1. Preparation before the test: 1 day before the test, each animal was numbered and weighed, and the hair on the test area was thoroughly clipped with an electric razor. Observe the health condition of each animal.
2. Intradermal induction: 0.1 mL was injected intradermally in pairs at the medial scapular site of the dehairing of each animal as shown in the figure below.
Site A: Inject a stabilizing emulsifier of Freund's Complete Adjuvant mixed with selected solvents in a 50:50 (v/v) ratio.
Site B: Test samples (undiluted extracts) were injected; control animals were injected only with the corresponding extract medium.
Site C: The test sample (the concentration used in site B) was mixed with the emulsifier prepared by Freund's complete adjuvant and the extraction medium (50%) at a volume ratio of 50:50 and then injected intradermal; The control group was injected with emulsifier prepared by blank liquid and adjuvant.
3. Local induction: 7d after intradermal induction, no irritation reaction was produced. 24h before the application of local dressing, the test area was pretreated with 10% sodium dodecyl sulfate, and after massaging and introducing into the skin, a sterile gauze with an area of 8cm2 was saturated with the extract, and locally applied on the medial side of the scapula of each guinea pig, covering the induced injection point. It was fixed with a closed bandage, and the bandage and gauze block were removed after 48h. Control animals were manipulated with blank leaching medium in the same way.
4. Stimulation: Fourteen days after local induction, a sterile gauze block was soaked in the test sample extract and control solution respectively and applied to the epigastric dehairing area (untested site during the induction phase) of each animal. It was fixed with a closed bandage and the bandage and dressing piece were removed after 24 hours ± 2 hours.
5. Observation: skin erythema and edema reactions are described and graded.
Closed application test (Beuhler)
1. Preparation for the test: 1 day before the test, each animal was numbered and weighed, and the hair on the test site was thoroughly clipped with an electric razor. Observe the health condition of each animal.
2. Induction: cut the samples into appropriate sized patches and applied them locally to the upper left dorsal region of each animal. Any closed bandage-like fixation and patch were removed after (6±0.5) h. The procedure was repeated for 3 consecutive days in 1 week and the same procedure was performed for 3 weeks. For control animals, a sterile gauze of appropriate size was soaked in control solution and applied to animals.
3. Excitation: (14±1) d after the last induction patch, test animals were excited with test samples. A patch was applied individually and locally to the untested part of each animal that was dehairing. The closed bandage and patch were removed after (6±0.5) h. The control animals were subjected to the same procedure.
4. Observation: Observe the skin reaction at the excitation site of the animals in the test group and control group at 24h and 48h after removing the dressing sheet, and describe and grade the skin erythematous and edematous reactions at each excitation site and at each observation time according to Magnusson and Kligman grading criteria.