This document discusses pyrogen testing methods. Pyrogens are fever-inducing substances, mainly lipopolysaccharides of bacterial origin. The rabbit pyrogen test, introduced in 1942, involves measuring temperature increases in rabbits injected with a test solution. If temperature increases exceed thresholds, the sample fails the test. The Limulus amebocyte lysate (LAL) test directly measures endotoxins using a lysate from horseshoe crab blood. Both tests are used to ensure medical products are free of pyrogens.

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.