05 Edition Chinese Pharmacopoeia Appendix Bacterial Endotoxin Thermogenic Aseptic Test English 1 10

Appendix Bacterial Endotoxin Thermogenic Aseptic Test

1. Scope

This test is used to detect the presence of bacterial endotoxins in injectable products. It is based on the principle that endotoxins from Gram-negative bacteria cause fever in rabbits, and the degree of fever is proportional to the amount of endotoxin present.

2. Principle

A sample of the product is injected into a group of rabbits. After a suitable interval, the rectal temperature of each rabbit is measured and recorded. The difference between the highest and lowest temperatures is calculated, and the mean temperature rise is determined. The test is repeated with a known amount of endotoxin added to the product. If the mean temperature rise of the test product is greater than the mean temperature rise of the control product, the test product is considered to contain endotoxin.

3. Apparatus

3.1. Syringe and needle: 5 mL syringe with a 21-gauge needle.

3.2. Thermometer: mercury-in-glass thermometer with a range of 35℃ to 43℃ and an accuracy of ±0.1℃.

3.3. Bath: water bath or heating block capable of maintaining a temperature of 37℃ ± 1℃.

4. Reagents

4.1. Product to be tested: the sample to be tested must be sterile and free of antimicrobial agents.

4.2. Endotoxin standard: a known amount of endotoxin standard (e.g. Escherichia coli endotoxin) is used to prepare a series of dilutions from 0.1 EU/mL to 10 EU/mL.

4.3. Control product: a product that is known to be free of endotoxin, such as sterile saline, is used as a control.

5. Procedure

5.1. Preparation of rabbits: rabbits weighing 2.0 kg to 3.0 kg are used. They are housed in individual cages and allowed to acclimate for at least 3 days before the test. The rabbits are fasted for 16 hours before the test.

5.2. Injection of test and control products: each rabbit is injected intravenously with 5 mL of the test product and 5 mL of the control product. The injections are given simultaneously in separate sites in the marginal ear vein.

5.3. Measurement of rectal temperature: the temperature of each rabbit is measured and recorded before injection (baseline) and at 1, 2, 3, 4, 5, and 6 hours after injection. The thermometer is inserted into the rectum to a depth of 2 cm, and the temperature is recorded to the nearest 0.1℃.

5.4. Calculation of mean temperature rise: the highest and lowest temperatures for each rabbit are identified, and the difference between them is calculated. The mean temperature rise for each group of rabbits is calculated by averaging the individual temperature rises.

5.5. Preparation of endotoxin-spiked product: a known amount of endotoxin standard is added to a sample of the product to be tested to give a concentration of 1 EU/mL.

5.6. Injection of endotoxin-spiked product: the procedure in steps 5.2 and 5.3 is repeated with the endotoxin-spiked product.

6. Interpretation of results

6.1. Calculation of the test result: the mean temperature rise for the test product and the control product is calculated. The difference between the two mean temperature rises is calculated, and the result is expressed in units of endotoxin per milliliter (EU/mL).

6.2. Acceptance criteria: the test product is considered to meet the requirements if the difference between the mean temperature rise of the test product and the mean temperature rise of the control product is less than the limit specified in the pharmacopoeial monograph for the product.

7. Precautions

7.1. Animal welfare: the use of animals in this test should be in accordance with the principles of animal welfare.

7.2. Endotoxin standard: the endotoxin standard used in this test must be calibrated against a reference standard.

7.3. Repeatability and reproducibility: the test must be repeated at least three times, and the results must be reproducible within acceptable limits.

7.4. Interference: some substances may interfere with the test and give false positive or false negative results. These substances should be identified and eliminated or their effects minimized.

7.5. Validation: the test method should be validated to demonstrate its suitability for the product being tested