Pyrogen testing

pyrogen testing

1. PYROGEN TESTING
Present by :- Guided by:-
Mr.N.P.Sawadadkar Prof.Tushar Thakur
Sir

2. Pyrogens
Pyrogens - fever inducing organic substances
 Responsible for many febrile reaction
 These are Endotoxin.
Having nature
Endogenous (inside body)
Exogenous (outside body)
Exogenous pyrogens –
mainly lipopolysaccharides
bacterial origin, but not necessary

3. Endotoxin characteristic
 thermostable
water-soluble
unaffected by the common bactericides
non-volatile
These are the reasons why pyrogens are
difficult to destroy once produced in a
product

4. Test for pyrogens = Rabbit test
the development of the test for pyrogens reach
in 1920
a pyrogen test was introduced into the USP
XII (1942)
The test consists of measuring the rise in body
temperature in healthy rabbits by the
intravenous injection of a sterile solution of
the substance under the test.

5. Why the Rabbit?
Reproducible pyrogenic response
Other species not predictable
Similar threshold pyrogenic response to
humans
 Rabbit chosen for economic purposes

6. Rabbit Pyrogen Test
Rabbits must be healthy and mature
New Zealand or Belgian Whites used mostly
Either sex may be used
Must be individually housed between 20 and
23°C
 Not varies more than ± 3º c.
 Free from disturbances likely to excite them.

7. equipment and material used in test (glassware,
syringes, needles etc)
 Must be free from Pyrogens by heating at 250º c
for not less then 30 minutes or any other method
retaining boxes (comfortable for rabbits as
possible)
Thermometers or thermistor probe (standardized
position in rectum, precision of ± 0.1°C)

9. Rabbit pyrogen test
Preliminary test (Sham Test)
intravenous injection of sterile pyrogen-free
saline solution
 Warm the pyrogen free solution up to 38.5ºc
to exclude any animal showing an unusual
response to the trauma (shock) of injection
any animal showing a temperature variation
greater than 0.6°C is not used in the main test

10. Rabbit pyrogen test -
main test:
group of 3 rabbits
preparation and injection of the product:
warming the product
dissolving or dilution
duration of injection: not more than 4 min
the injected volume: not less than 0.5 ml per 1 kg and not
more than 10 ml per kg of body mass
determination of the initial and maximum temperature
all rabbits should have initial Temperature: from 38.0 to
39.8°C
the differences in initial Temperature should not differ from
one another by more than 1°C

11. Interpretation of the results:
the test is carried out on the first group of 3 rabbits; if
necessary on further groups of 3 rabbits to a total of 4
groups, depending on the results obtained
intervals of passing or failing of products are on the
basis of summed temperature response

12. The result of pyrogen test:
No.of Rabbits Individual
Tempt. rise
(°c)
Tempt.
Rise in
group (°c)
Test
3 rabbits 0.6 1.4 Passes
If above not passes
3+5 = 8 rabbits
0.6 3.7 Passes
If above test not passes the sample is said to pyrogenic.

13. LAL Test
Limulus amebocyte lysate test.
 to measure the concentration of endotoxins of
gram-negative bacterial origin
reagent: amoebocyte lysate from horseshoe
crab, Limulus polyphemus

14. Limulus polyphemus = horseshoe crab

15. Principle
The addition of solution containing endotoxin
to a solution of lysate produce turbidity.
The rate of reaction depends upon
concentration of endotoxin , the pH and the
temperature.
The endotoxin reference standard is the freeze
dried.
The test is based on the primitive blood-
clotting mechanism of the horseshoe crab

16. Commercially derived LAL reagents
bleeding adult crabs into an anticlotting solution
washing and centrifuging to collect the amebocyte
lysing in 3% NaCl
lysate is washed and lyophilized for storage
 activity varies on a seasonal basis and
standardization is necessary.

17. Test performance (short)
avoid endotoxin contamination
Before the test:
interfering factors should not be present
equipment should be depyrogenated

the sensitivity of the lysate should be known
Test:
equal volume of LAL reagent and test solution (usually 0.1
ml of each) are mixed in a depyrogenated test-tube
incubation at 37°C, 1 hour
remove the tube - invert in one smooth motion (180°) - read
(observe) the result

18. Endotoxin concentration monitoring
Following method are used to monitor the endotoxin
concentration
Method A: El-clot method: limit test
Method B: semi-quantitative gel-clot method
Method C: kinetic turbidimetric method
Method D: kinetic chromogenic method
Method E: end-point chromomeric method

19.  different techniques:
the gel-clot technique - gel formation
the turbidimetric technique - the development
of turbidity after cleavage of an endogenous
substrate
the chromogenic technique - the development
of color after cleavage of a synthetic peptide-
chromogen complex

20. REFERENCES
 U.S.PHARMACOPEIA
 Pharmaceutical Formulations by
M.E.Aulton, H.C. Ansel.page no 195-196.