Protocol for Abnormal toxicity test for Rubber Closures of Parenteral Products
1. Name of the study: Abnormal Toxicity
2. Animal species used: Male/Female- Mice and Rabbit
3. Criteria for choosing the animal:
Ø Mice:
a. Age: Adult
b. Body weight: 17 to 22 g.
Ø Rabbit:
a. Age: Adult
b. Body weight: 2.0 to 3.0 kg.
*Note: The females should be nulliparous and non-pregnant. If the requirement will arise, the number can be increased by the number of animals scheduled for the completion of the study.
4. Housing procedure: Animals should be caged individually. They have housed in a polypropylene cage in air–condition area at 22 C (+ 3o) for rodents and 20oC (+ 3o) for rabbits, and the relative humidity is 30 to 70 % with 12 hr light & dark cycle. All the animals had free access to a standard pellet diet clean water ad libitum.
5. Duration of study: 72 hours (3 days for observation of toxic effects)
6. Route of administration: intravenous
7. Instrument used: Precision weighing balance, Screw capped 200ml bottles, Beakers. Measuring cylinder, Pipette, Oven, Autoclave, Needle syringe.
8. Methodology:
Standard drug: Take the standard drug as per requirement.
Preparation of test drug:
Place a selected number of intact closures in an extraction container, add 50ml 0f sterile water for injection, cap, and agitate for 2 to 3 minutes. Decant using a stainless steel screen to hold the closures with the sample and caps in an oven at a temperature of about 50° and allow drying for not more than 16 hrs.
Place two properly prepared samples to be tested in separate extraction containers and add to each container 1ml per 1.25 ± 0.1 cm2, of sterile normal saline solution and extract by heating in an autoclave at 121°± 0.1° for 60 minutes. Allow adequate time for the liquid within the container to reach room temperature but not below 20°. Agitate vigorously for several minutes and then aseptically transfer the extract immediately to dry sterile containers. Carry out the following tests omitting the closures, within 24 hrs.
Test A. Agitate each extract vigorously prior to the withdrawal of injection doses to ensure the even distribution of the extracted matter. However, the visible particulate matter should not be injected intravenously.
Inject intravenously 1.0 ml of each of the sample extracts and the blank solution into each of the five healthy albino mice weighing between 17g to 22g. The mice should not have been used previously. Observe the animal immediately after injection and then at least at 24, 48, and 72 hrs. If during this period none of the animals treated with the sample extracts shows a significantly greater reaction than the animals treated with blank solution, the sample passes the test. If any animal treated with the sample extracts shows only slight signs of toxicity, and not more than one animal shows gross symptoms of toxicity or death, repeat the test using ten mice for each extract shows no significant reaction greater than that seen in the animals treated with the blank.
Test B. Agitate each extract vigorously prior to the withdrawal of injection doses to ensure the even distribution of the extracted matter.
Select healthy, thin-skinned albino rabbits whose fur can be closely clipped and whose skin is free from mechanical irritation or trauma. Rabbits previously used in unrelated tests, such as the test for pyrogens, and that have received the prescribed rest period, may be used provided they have clean, unblemished skin. In handling the animals, avoid touching the injection sites when the animals are observed. Use two animals for each extract and inject each into five sites of each animal. Inject into each animal intracutaneously 200 µl of the sample extract and the blank, using one side of the animal for the sample extract and the other for the blank.
On the day of the test, closely clip the fur on the animal’s back on both sides of the spinal column over a sufficiently large test area. Avoid mechanical irritation and trauma. Remove loose hair by means of a vacuum. If necessary, swab the skin lightly with diluted ethanol, and dry the skin prior to injection. During these periods examine the injection sites for evidence of any tissue reaction such as erythema, edema, and necrosis.
If each animal at any observation period shows a reaction to the sample extract that is not significantly greater than that to the blank, the sample meets the requirements of the test. If during any observation period, the reaction to the sample extract is questionably greater than that to the blank, repeat the test using three additional rabbits. In the repeat test, the reaction to the sample extract in any of the three animals is not significantly greater than that to the blank.
a. Experiments: Animals are divided into 2 groups-
For Mice-
Test group: 5 animals
Control group: 5 animals
For Rabbits-
Test group: 1 animal
Control group: 1animal
9. Calculations:
10. Dose selected
a. 1ml for each animal for the test group
b. 1ml for each animal for the control group
11. The total number of animals used for the study: 10 for mice & 2 for rabbits.
12. Total number of groups: 02
13. Euthanasia (If applicable): not required
Reference: Indian Pharmacopoeia 2018 (p.101-107).
END OF DOCUMENTS
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