The test for Abnormal / Undue Toxicity / Safety test is provided to determine compliance with the requirements given in individual monograph / specifications.
This test is intended to detect any unexpected, unacceptable, biological reactivity in a substance. This is an invitro test designed for safety assessment of biologics and biotechnology derived products.
1. Keep the animals individually in a quiet area with an appropriate uniform temperature allowing a variation of ± 2 OC and uniform humidity and free from disturbance.
2. Carry out the test in a quiet room where there is no risk of disturbance which can excite the animals and in which the room temperature is within 3 OC of that of their living quarters or in which the animals have been kept for at least 18 h before the test.
3. Withhold food from the animals overnight and until the test is completed , with hold water only during the test.
TEST FOR UNDUE TOXICITY ( As per USP )
1. Inject intravenously into each of 5 healthy mice, weighing between 17 - 22 g the quantity of the substance to be examined prescribed in the individual monograph, dissolved in 0.5 mL of water for injection should normally occupy about 15 - 30 sec, unless otherwise prescribed in the individual monograph.
2. The substance passes the test if none of the mice dies within 24 h or within such time as is specified in the individual monograph.
3. If one of the animals dies, repeat the test. The substance passes the test if none of the animals in the second group dies within the time interval specified.
TEST FOR ABNORMAL TOXICITY (As per BP)
Use Method - A unless otherwise directed.
Where under the heading Abnormal Toxicity in an individual monograph it states that the substance being examined is to be dissolved in Albumin solution, this reagent may be replaced by a solution in water for injections containing an appropriate amount of a suitable protein that has been shown to be pyrogen-free and also shown, by a suitable method, to be free from proteolytic activity. The solution is adjusted to an appropriate pH.
1. Inject intravenously into each of five mice the quantity of the substance being examined prescribed in the monograph, dissolve in 0.5 mL of water for injection or Sodium Chloride injection unless otherwise prescribed in the monograph.
2. The injection should normally be administered over a period of 15 to 30 sec., unless otherwise prescribed in the monograph.
3. None of the mice should die within 24 h, or within such time as is specified in the monograph.
4. If one of the animals dies within the specified time, repeat the test. None of the animals in the second group should die within the specified time.
1. Unless otherwise prescribed in the monograph inject one dose but not more than 1.0 mL intraperitoneally into each of 5 mice and one dose but not more than 5.0 mL intraperitoneally into each of two guinea-pigs.
2. None of the animals should die or show of ill health within 7 days.
3. If one of the animals dies or shows signs of ill health within 7 days, repeat the test. None of the animals in the second group should die or shows signs of ill health within 7 days.
SAFETY TEST ( As per USP)
1. Select five healthy mice not previously used for testing, weighing between 17 - 22 g, unless otherwise specified in the individual monograph, and maintained on an adequate balanced diet.
2. Prepare a test solution as directed in the individual monograph. Unless otherwise directed in the individual monograph, inject intravenously a dose of 0.5 mL of the test solution into each of the mice, using a 26 gauge needle of suitable length.
3. Observe the animals over the 48 h. following the injection.
4. If, at the end of 48 h., all of the animals survive and not more than one of the animals shows outward symptoms of a reaction not normally expected of the level of toxicity related to the article, the requirements of this test are met.
5. If one or more animals die or if more than one of the animals shows signs of abnormal or untoward toxicity of the article under test, repeat the test using at least another 10 mice similar to those used in the initial test, but weighing 20 ± 1 g. In either case, if all of the animals survive for 48 h. and show no symptoms of a reaction indicative of an abnormal or undue level of toxicity of the article, the requirements of the test are met.
1. For biologics, perform the test according to the procedures prescribed in the Federal regulations (see Biologics , Section 610.11), using not less than 2 mice similar to those described above but weighing less than 22 g and not less than 2 healthy guinea - pigs weighing less than 400 g.
2. For a liquid product or a freeze-dried product that has been constituted as directed in the labeling, inject a volume of 0.5 mL intraperitoneally into each mouse, and inject a volume of 5.0 mL intaperitoneally into each guinea-pig.
3. For freeze-dried products for which the volume of constitution is not indicated in the label, or for non liquid products other than freeze-dried products, perform the test using the route of administration, test dose, and diligent approved by the Center for Biologics Evaluation and Research (FDA) on the basis of substantial evidence demonstrating that the test variation will assure sensitivity equal to or greater than that of the test described above.
1. Observe the animals for a minimum observation period of 7 days.
2. If all of the animals survive the test period, do not exhibit any response that is not specific for or expected from the product and that may indicate a difference in such product quality, and weigh no less at the end of, the test period than at the time of injection, the requirements of the test are met.
3. If the article fails to meet the requirements, the test may be repeated as in the initial test, in the one or both species in which the requirements were not met .
4. If the animals fulfill the criteria specified for the initial test, the article meets the requirements of the test. If the article fails to meet the requirements after the first repeat test, and not less than 50 % of the total number of animals of the species in which the requirements of the test were not met in the combined initial and first retests have survived, a second retest may be performed.
5. Use twice the number of animals of the relevant species used in the initial test. If the animals fulfill the criteria specified for the initial test, the requirements of the test are met.
Note: Use the method as per the Pharmacopoeial status (grade) of material. In case of In-house Specifications, follow the method as per IP or as specified.