BACKGROUND
Pyrogens are the fever inducing organic substances (metabolic products of microorganisms) responsible for many febrile reactions. Pyrogen testing should be done to every batch of pharmaceutical product (particularly parenterals) for which water is the usual vehicle.1
The best animal model for pyrogen test is the rabbit as it generates reproducible results that are similar to threshold response to humans and also economic.2
REQUIREMENTS
Animals: Healthy matured rabbits of either sex
Instruments used: Pyrogen free syringes, needles, glass ware, clinical thermometer or thermistor probe
Reagents: 0.9% sodium chloride injection as diluent
PROCEDURE
Select healthy matured rabbits of either sex for the study. House them individually in the place that is free from disturbances that likely to excite them and maintain the room temperature at 20-230c. Make all the materials and equipment pyrogen free either by heating the same at 2500c for not less than 30 minutes or any other method.
Standardized clinical thermometer with precision of ±0.10c is used to measure rectal temperature of the rabbit. Test them to determine that maximum reading is attaining in <5 minutes or not.
Pyrogens testing of solutions should done in two steps.3
a. Preliminary test (Sham test)
Conduct Sham test for animals using for first time in pyrogen testing or have not been used during the two previous weeks. Hence acclimatize them for 1-3 days before using for pyrogen testing of sample. Select three rabbits and fast the rabbits overnight with free access to water and withhold water during the test.
Inject sterile pyrogen free saline solution intravenously at a dose of 10 ml per kg of body weight. Record the temperature of animals 90 minutes prior to the injection and continue for 3 hours at an interval of half an hour after injection. Exclude any animal showing temperature variation of 0.6 or more for main test.
b. Main test
Select three rabbits that passed the Sham test. Determine the initial body temperature of the rabbits and it should be between 38-39.80c. Dilute Solution to test with pyrogen free saline solution or any solution prescribed in monograph.
Warm the test liquid to 38.50c before injection. Inject the test solution to the animal slowly into the marginal vein of the ear for a period of not more than 4 minutes and the volume injected should not less than 0.5 ml/kg and not more than 10 ml/kg of body mass. Determine the temperature of each animal at every half an hour for 3 hours after injection.
Interpretation of the result
Consider any decrease in final body temperature as rise in zero. Consider the test solution as pyrogen free if the response in individual rabbit showing a temperature less than 0.60c or if the sum of responses of three rabbits does not exceed 1.40c.
If the temperature exceeds than the above, then continue the test using other five rabbits. If not more than 3 rabbits out of eight shows individual rise in body temperature of 0.60c or if the sum of the responses of the group of 8 rabbits does not exceed 3.70c then the preparation under test is passed and considers it as pyrogen free.
OBSERVATION
Table 1: Recording of initial and final body temperatures.
|
Number of rabbits |
Initial body temperature (in 0c) |
Final body temperature (in 0c) |
|||||
|
30 min |
60 min |
120 min |
180 min |
240 min |
300 min |
||
|
1st animal |
|||||||
|
2nd animal |
|||||||
|
3rd animal |
|||||||
Table 2: Result of pyrogen test.
|
No. of rabbits |
Individual body temperature raise (in 0c) |
Body temperature rise in group (in 0c) |
Test |
|
1st animal |
Pass/fail |
||
|
2nd animal |
|||
|
3rd animal |
CONCLUSION
Difference in initial and final body temperature of rabbits individually or in group determines the presence or absence of pyrogens in a test substance.
REFERENCES
- Jayanthi VR. Pyrogens. Available at: http://www.pharmainfo.net/pyrogens. Accessed on 07 October 2016.
- Sawadadkar NP, Thakur T. Pyrogen testing. Available at: https://www.slideshare.net/nilesh1208/pyrogen-testing. Accessed on 06 October 2016.
- Pyrogen test. Available at: https://teachntest.files.wordpress.com/2016/08/pyrogen-test.pdf. Accessed on 10 October 2016.
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- Study of local anesthetics by Infiltration anaesthesia in guinea pig
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