Irritation Test
Skin Irritation Test
Skin Irritation Test： It is an experimental method to assess the skin irritation of substances, mainly used to detect the skin irritation reaction of cosmetics, pharmaceuticals, chemical substances and so on. The test evaluates the skin irritation of a substance by observing the reaction of the skin of experimental animals or human beings after contact with the substance, and provides a basis for product safety and risk assessment.
Test methods:
1. Preparation before the test:
4h ~ 24h before the test, the animals were marked and weighed, and the hair was removed from both sides of the spine on the back of the animals (about 10cm×15cm), which was used as the test and observation site.
2. Test procedure:
Apply the test material to both sides of the spine on the back of the animal, and apply the negative control material to the control site in the same way. The dressing was fixed with a bandage for at least 4 h. At the end of the contact period, the dressing was removed, the contact site was labeled with persistent ink, and the residual test material was removed with lukewarm water. The erythematous and edematous reactions at the contact site were observed and recorded at 24h, 48h and 72h after exposure.
3. Results:
After 72h scoring, all erythema and edema scores induced at (24±2) h, (48±2) h, and (72±2) h, respectively, for the test sample of each animal were summed, and then the primary irritation index of a particular animal was calculated by dividing the sum of all scores by 6 (two test/observation sites, 3 time points). The primary irritation index for a test sample was calculated by adding all primary irritation scores for each animal and dividing by the total number of animals (3). A control primary irritation score was calculated in the same way and subtracted from the test material primary irritation score to obtain the primary irritation score.
Oral irritation test
Oral irritation test：should be considered only for materials intended to come into contact with oral tissues and only when safety data cannot be obtained by other methods, to assess the potential of the material to produce an oral tissue irritation response under the conditions of the test.
Test methods:
1. Preparation before the test:
Screened animals were examined for abnormalities after removing the animal's collar and flipping the buccal sac to rinse it with saline.
2. Test procedure:
A φ10mm cotton ball was soaked with 0.5mL of test solution and placed into one side of the animal's cheek pouch. No sample was placed in the other cheek pouch, and two groups of gophers were taken as negative control groups according to the different extraction media. Keep the cotton ball in contact with the oral mucosa of the gopher for 5 min. After contact, remove the collar and cotton ball, rinse the cheek pouch with saline, and re-collar the animal back to the cage. Repeat the above procedure once every hour for 4 hours. The buccal sacs were observed visually after removal of the test balls and were examined before each contact (if multiple contacts were required) and scored according to the following scoring system to determine the surface erythema reaction of the buccal sacs for each observation period.
The buccal sacs were observed visually after 24h of exposure and the animals were painlessly sacrificed. The mucosa and surrounding tissues at the contact site of the specimen were taken, fixed in 10% formalin, sectioned by paraffin embedding, and prepared by HE staining for histological evaluation.
3. Results:
Evaluation by visual observation: Visual observation of the mucous membrane at the specimen placement site during each observation period. Comparing the mucosa of the test side and the control side, the clinical performance of the oral mucosa was scored according to the Oral Mucosal Response Scoring System. The observation scores of each animal in each observation period were summed up and divided by the total number of animals observed to obtain the average score of each animal.
Rectal stimulation test
Rectal stimulation test：Only materials intended for use in contact with rectal tissue should be considered, and only if safety data cannot be obtained by other methods. The potential of the material to produce a rectal tissue irritant response under the test conditions should be evaluated.
Test methods:
1. Procedure
A short length of flexible tubing (6 cm) or a blunt-tipped cannula is connected to a syringe with a capacity greater than 1 mL. The syringe and catheter are filled to the point where the animal can receive 1 mL of the test sample. A separate set of syringes and connecting catheters should be prepared for each animal. The animal should be immobilized in an immobilizer to facilitate access to the perineal area. Alternatively, the animal may be restrained with an immobilizer and then the hind limbs immobilized to expose the perineum. The catheter may be moistened with a control sample or appropriate lubricant prior to insertion. The perineum was exposed by lifting the animal's tail, and the moistened catheter was gently inserted into the rectum and 1 mL of test solution was injected with a syringe. The catheter was withdrawn and disposed of in an appropriate manner. Due to the variability of rectal volume in individual animals, the test sample may be spilled during or after injection, and the spilled liquid can be gently wiped away with soft paper towels. Repeat the above procedure at intervals of (24±2) h for 5 d. For long-term repeated exposure tests, the number, duration and intervals of exposure should be determined according to the expected duration of clinical exposure.
2. Observation
The perineal discharge, erythema, and irritation were noted and recorded at (24±2)h after initial exposure and before each test. Animals that had excessive discharge, swelling, or were difficult to administer should be painlessly put to death for histological examination.
3. Results
Animals were painlessly sacrificed (24±2) h after the last exposure, and the rectum was dissected longitudinally after complete excision to examine the epithelial tissue layer for irritation, damage, and necrosis. The ends of rectum and large intestine were fixed in appropriate fixative and then examined histologically. The rectal tissues of the test rabbits were compared with those of the control rabbits. The condition of the rectal tissue of each animal under visual observation was recorded and described, noting the differences between the test and control sites.
4. Histological evaluation
The stimulatory effect on the rectal tissue should be evaluated by a pathologist. The microscopic evaluation scores of all animals in the test group should be added together and divided by the total number of observations to obtain the average score for the test group. The maximum score is 16. The control group was calculated in the same way. If the total microscopic evaluation score of the control animals was greater than 9, it indicated that the test operation might have caused injury. If other tests or control animals show similarly high scores, retesting may be necessary. The stimulus index was calculated by subtracting the mean score of the control group from the mean score of the test group.
Vaginal irritation test
Vaginal irritation test：should be considered only for materials intended to come into contact with vaginal tissue and only when safety data cannot be obtained by other methods. The potential of the material to produce a vaginal tissue irritation response under the test conditions is assessed.
Test methods:
1. Preparation before the test:
New Zealand rabbits were taken and randomly divided into test and control groups of 3 animals each. The animals were immobilized in a fixator to expose the vaginal opening. The catheter was wetted and treated with control sample or appropriate lubricant before insertion.
2. Procedure:
The vaginal opening was exposed by lifting the animal's tail, and then a moistened catheter was gently inserted into the vagina and 1 mL of test solution was injected with a syringe attached to a flexible catheter. The procedure was repeated at 24-h intervals for 5 d. Observations of vaginal and perineal overflow, erythema, and irritation were made 24 h after the initial exposure and prior to each test procedure. Twenty-four hours after the final exposure, the animals were painlessly sacrificed, and the vagina was completely excised longitudinally for gross examination of the epithelial tissue layers for irritation, damage, and necrosis.
Histological examination: After the vaginal tissues were fixed in formalin, the anterior 2cm, central 2cm and terminal 2cm of the vagina were sampled and marked as 1, 2 and 3, respectively, and the thickness of the samples was about 0.3cm, one section at each place, near the anterior end of the vagina towards the cut surface, and then the tissues were subjected to paraffin embedded sectioning, and HE stained sectioning, and the histological evaluation was carried out. The vaginal tissues of test rabbits were compared with those of control rabbits. The condition of the vaginal tissue of each animal under visual observation was recorded and described.
3. Results:
Naked eye observation: The vaginal tissues were completely excised and examined for irritation, damage and necrosis of the epithelial tissue layer. The vaginal tissues of the test rabbits were compared with those of the control rabbits, and the condition of the vaginal tissues of each animal under visual observation was recorded and described.
Histological observations: Each tissue was scored according to the following scoring system. The average score of the test group was obtained by adding the scores of the microscopic evaluations of the animals in the test group and dividing by the total number of observations. The average score of the test group was subtracted from the average score of the control group to obtain the stimulation index.