Expt No 21 Pyrogen Testing

Summary

This video demonstrates the official method for testing pyrogens in injectable solutions using rabbits. The test involves monitoring the fever response in three rabbits after intravenous injection of the test sample. Key parameters include measuring temperature changes at regular intervals, ensuring that individual temperature rises do not exceed 0.6°C, and the total rise for all three rabbits stays below 1.4°C. If these conditions are not met, the test is repeated with additional rabbits. The procedure ensures that injectable preparations are free from pyrogens, which can cause harmful fever responses in humans.

Takeaways

• 😀 **Pyrogens** are high molecular weight lipopolysaccharides, often endotoxins, that cause fever when introduced into the body, making it crucial to test injectable preparations for their presence.
• 😀 **Endotoxins** are heat-stable toxins found in bacterial cell walls and are a significant cause of pyrexia (fever) when they contaminate injectable solutions.
• 😀 The **official test for pyrogens** involves measuring the rise in body temperature in rabbits after injecting a test solution, ensuring the solution is free from pyrogen contamination.
• 😀 The test uses **three healthy rabbits** (weighing at least 1.5 kg) to monitor temperature changes, and results are considered valid if specific conditions are met.
• 😀 **Temperature readings** are taken every 30 minutes over a span of 3 hours, and any significant temperature rise must be carefully interpreted to assess pyrogen contamination.
• 😀 The **marginal ear vein** of the rabbit is used for intravenous injection of the test solution at a temperature of 37°C ± 2°C.
• 😀 If the **temperature rise** in any rabbit exceeds 0.6°C, the test is invalid, and the procedure must be repeated with new animals.
• 😀 The **sum of the temperature responses** for all three rabbits should not exceed 1.4°C for the test to pass, ensuring the test sample is pyrogen-free.
• 😀 If the initial test fails, a follow-up test with **five additional rabbits** is conducted, and the total temperature rise from all eight rabbits should not exceed 3.7°C.
• 😀 The **Taylor thermometer with six probes** is used to monitor the temperature of multiple rabbits simultaneously, with an accuracy of 0.1°C, ensuring precise readings during the experiment.

Q & A

What is the purpose of the pyrogen test described in the script?

The purpose of the pyrogen test is to observe and study the effect of pyrogens in rabbits, specifically testing the presence of pyrogens in injectable preparations. The test helps ensure that these preparations are free from pyrogens, which could cause fever or other adverse reactions in humans.

What are pyrogens and why are they a concern in injectable preparations?

Pyrogens are high molecular weight lipopolysaccharides, which are chemical compounds made up of lipid and carbohydrate components. They are a concern in injectable preparations because they are endotoxins that can cause fever and other inflammatory responses when introduced into the body.

What are the main objectives of the pyrogen test as mentioned in the transcript?

The main objectives include understanding the nature of pyrogens, how they produce their effects, and how pyrogen testing is conducted in both laboratories and industry. It also aims to ensure that injectable solutions are free from pyrogens using official testing methods like the rabbit fever response test.

What are the key requirements for conducting the pyrogen test?

The key requirements include using healthy rabbits weighing at least 1.5 kg, sterile glassware and syringes, water for injections, pyrogen-free diluent solutions, and a controlled environment. The sample being tested should be prepared and injected in specific conditions, such as maintaining a temperature of 37°C for the test solution.

How is the pyrogen test performed using rabbits?

The test is performed by injecting the test sample into the marginal ear vein of the rabbit. Temperature is recorded at regular intervals (every 30 minutes for 3 hours) using a thermometer with six probes, which are inserted into the rectum of the rabbits to monitor their body temperature for any fever response.

What are the conditions for the pyrogen test to pass as per the official guidelines?

The test passes if the temperature rise in any rabbit is less than 0.6°C and the sum of the temperature rises in the three rabbits does not exceed 1.4°C. If these conditions are not met, additional rabbits are tested until the sum of the temperature rises from eight rabbits does not exceed 3.7°C, and no more than three rabbits show a temperature rise greater than 0.6°C.

What happens if the initial test with three rabbits fails?

If the initial test fails, a second round of testing with five additional rabbits is performed. The test passes if no more than three rabbits show a temperature rise greater than 0.6°C, and the sum of the temperature rises of all eight rabbits is less than 3.7°C.

What instrument is used to measure temperature during the pyrogen test?

A six-probe tail thermometer is used to measure the temperature of the rabbits during the test. This instrument allows for simultaneous temperature readings from up to six rabbits, ensuring accuracy and efficiency in monitoring the fever response.

Why is it important to ensure that the rabbits are not stressed or unhealthy before the test?

It is important because stress or health issues in rabbits could skew the temperature readings, leading to inaccurate results. Only healthy rabbits within the specified temperature range should be used to ensure the reliability of the test.

What are endotoxins, and why are they particularly relevant to pyrogen testing?

Endotoxins are heat-stable toxins present in the outer membrane of certain bacteria. They are a major component of pyrogens and can induce fever in animals and humans. Because endotoxins are not easily removed and can remain active for long periods, it is critical to ensure that injectable solutions are free from endotoxins during manufacturing.