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)
8.
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
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
Monkeys, horses, dogs, cats, and rabbit have reproducible responses
Rats, guinea pigs, mice, hamsters, chicks, etc, are irregular and unpredictable
Rabbit and dog chosen chosen for economic purposes, BUT…
Rabbit has labile thermoregulatory process, and is susceptible to false positives. A negative test is more significant than a positive one. The dog has a more stable thermoregulatory system, and is less sensitive to pyrogen. Therefore a positive is more significant than a negative one.
Similar threshold pyrogenic response to humans. HOWEVER, as dose is increased, humans respond more vigorously.
USP XX had only two requirements for the animals, that they be healthy and mature.
NZ or Belgian Whites are mostly used
Either sex may be used, but kept to a single sex to avoid outside stimuli