Importance of Rabbit and LAL Test Pyrogen Test in Parenterals

What are Pyrogens?

Pyrogens are fever-inducing substances usually derived from microorganisms, and when present systemically in sufficient quantity, may lead to severe signs of inflammation, shock, multiorgan failure, and sometimes even death in humans.

Types of Pyrogen Tests?

The rabbit pyrogen test (RPT) and the Limulus amebocyte lysate (LAL) test have traditionally been used to determine pyrogenicity.
It is important to conduct both of these tests for parenteral products like vaccines
In keeping with the policy of the 3Rs, the FDA has accepted the use of the in vitro test in lieu of the animal test for more than 30 years.
The LAL test is a highly quantitative way to measure the endotoxin levels of gram-negative bacteria and is based on the clotting reaction of the horseshoe crab’s hemolymph.
WHO guidelines recommend that developers provide evidence that their vaccine formulation doesn’t interfere with the LAL test.

Pyrogen Test

A qualitative biologic test based on rabbit fever reaction.
Rabbits show a physiologic response to pyrogens similar to that of human beings.
A greater danger exists from the injection of LVP containing pyrogens than from SVP.
IM Injection has a lower pyrogenic effect than IV injection.
The rise in body temperature of rabbits after intravenous injection of a sterile solution of the substance under investigation is measured in this test. It’s for items that can be given intravenously in a dose of no more than 10 ml per kg in a time of no more than 10 minutes and tolerated by the test rabbit.

Preliminary Test (SHAM test)

If animals are used for the first time in a pyrogen test or have not been used during the two previous weeks, condition them one to three days before testing the substance being examined, by injecting I.V. 10 mL of pyrogen-free saline solution per kilogram of body weight.
Carry out the test in a room with no risk of disturbing or exciting the animals, and a room temperature that is within 3 degrees of the area where the animals are housed, or in which the animals have been maintained for at least 18 hours before the test.
Food and drink should be withheld from the animals overnight and until the test is completed.
Record the temperature of the animals beginning at least 90 min. before injection and for 3 hours following injection of the solution under investigation.
Any animal showing a temp. In the main test, any change of 0.6 or greater must be avoided.

Main test

Use a group of three rabbits to do the experiment.

Preparation of the sample

Dissolve the substance under investigation in pyrogen-free saline solution or another solution specified in the monograph, or dilute it with it. Warm the liquid under examination to approximately 38.5ºC before injection.

Procedure

Inject the solution(38.5ºC) under examination slowly into the marginal vein of the ear of each rabbit over a period not exceeding 4 minutes, unless otherwise prescribed in the monograph.
Record the temperature of each animal at half-hourly intervals for 3 hours after the injection.
The difference between the “starting temperature” and the “maximum temperature,” which is the greatest temperature a rabbit has ever been measured at, is considered its response.
The volume of sample to be injected depends on the preparation being tested and is specified in the particular monograph.
The injection volume must be between 0.5 and 10 milliliters per kilogram of body weight.

Interpretation of results (IP)

If the sum of the responses of the group of three rabbits does not exceed 1.4°C and if the response of individual rabbits is less than 0.6°C, the preparation under examination passes the test.
If it exceeds, repeat the test with five more rabbits.
If not more than three of the eight rabbits show individual responses of 0.6°C or more, and if the sum of responses of the group of eight rabbits does not exceed 3.7°C, the preparation under examination passes the test.

Attempt: Parenterals MCQ Test

Limulus Amoebocyte Lysate (LAL) Test

Limulus amoebocyte lysate (LAL) is a test that’s simple to do and can be very helpful in the detection of pyrogens.
Certain bacterial cell-wall lipopolysaccharides (i.e. endotoxins) lead to gelation of blood cell (amoebocyte) lysates of the Limulus polyphemus crab.
The reagent is mixed with equal parts serially diluted test sample. The mixture is checked for evidence of a clotting reaction (gel clot) after incubation at 37°C, and the test samples are compared to parallel dilutions of a reference endotoxin.

The presence of bacterial endotoxins in the sample is indicated by the formation of a gel clot, and the test is considered positive.

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