PYROGEN TESTING

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

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

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

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.

Why the Rabbit?

Reproducible pyrogenic response Other species not predictable Similar threshold pyrogenic response to

humans Rabbit chosen for economic purposes

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.

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)

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 0.6C is not used in the main test

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 39.8C

the differences in initial Temperature should not differ from one another by more than 1C

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

The result of pyrogen test:

No.of Rabbits Individual Tempt. rise (°c)

Tempt. Rise in group (°c)

Test

3 rabbits 0.6 1.4 PassesIf above not

passes 3+5 = 8 rabbits

0.6 3.7 Passes

If above test not passes the sample is said to pyrogenic.

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

Limulus polyphemus = horseshoe crab

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

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.

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

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

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

REFERENCES

U.S.PHARMACOPEIA Pharmaceutical Formulations by

M.E.Aulton, H.C. Ansel.page no 195-196.