PYROGEN TEST

This test consists of measuring the rise in body temperature evoked in rabbits by the injection of a sterile solution of the substance being examined.

PYROGENS:

Pyrogens are the by-products of microorganisms mainly of bacteria, molds and viruses.

During the processing these pyrogens may come from water, active constituent or the excipient or from the equipments. Chemically these pyrogens are lipid substances associated with carrier usually polysaccharides or may be proteins.

Parenteral solutions are officially tested for the presence of pyrogens by a biological test in which “FEVER” response of rabbits is used as criteria.

SELECTION OF ANIMALS:

Use healthy adult rabbits of any sex weighing not less than 1.5kg.

Feed them a well balanced diet not containing any antibiotics during one week preceding the test.

A rabbit should not be used in the pyrogen test if:

It has been used in a negative pyrogen test in the preceding three days or

It has been used in the preceding three weeks in a pyrogen test in which the substance under examination fails to pass the test or

It has been used at any time in the pyrogen test in which the mean response of the rabbit in the group exceeds 1.2C.

MATERIALS NEEDED:

(a). THERMOMETERS:

The thermometer or electrical device used should indicate the temperature with a great sensitivity and should be inserted in the rectum of the rabbit to the depth of about 5.2cm (B.P specification) or 7.2cm (USP specification). The depth of insertion is constant for any rabbit in every group.

When an electrical device is used, it should be inserted in the rectum of the rabbit 90 minutes before injection of the solution to be examined and left in position throughout the test.

(b). GLASSWARE, SYRINGES AND NEEDLES:

All the glassware, syringes and needles must be thoroughly washed with water and heated in a hot air oven at 250C for 30 minutes or at 200C for an hour.

(c). RETAINING BOXES:

The retaining boxes for rabbit in which the temperature is being measured by and electrical device should be made in such a way that the animals are retained only by loosely fitting neck stocks, the rest of the body remains relatively free, so the rabbit may sit in a normal position.

The animals must be put in box not less than one hour before the test and remain there throughout the test.

PRELIMINARY TEST (SHAM TEST):

One of the three days before testing the product, inject pyrogen free isotonic NaCl solution (10ml/kg body weight warmed at 38.5C intravenously) into animal, which has not been used during the two previous weeks.

Record the temperature of animal beginning at least 90 minutes before injection and continuing for 3 hours after injection of solution.

Any animal showing a temperature difference greater than 0.6C must not be used in the main test.

MAIN TEST:

Carry out the test using a group of three rabbits.

PREPARATION AND INJECTION OF SAMPLE

Warm the liquids to be examined to approximately 38.5C before injection. The sample liquid to be injected may be diluted with a pyrogen free isotonic NaCl solution.

Inject the solution slowly into the marginal vein of the ear of each rabbit over a period of four minutes, unless otherwise mentioned in the monograph.

The volume of the injection should be not less than 0.5ml/kg body weight and should not be more than 10ml/kg body weight.

DETERMINATION OF INITIAL AND MAXIMUM TEMPERATURE

The initial temperature of each rabbit is the mean of two temperature readings, recorded for that rabbit at an interval of 30 minutes immediately preceding the injection.

While the maximum temperature is the highest temperature recorded for that rabbit three hours after the injection of the preparation being tested.

Record the temperature of each animal at an interval of 30 minutes beginning at least 90 minutes before the injection.

The difference between the initial temperature and the maximum temperature of each rabbit is taken to be its response.

When this difference is negative, the result is counted as zero response.

REJECT THE RABBIT IF:

It is having an initial temperature higher than 39.8C or lower than 38.0C.

It is showing temperature difference more than 0.2C between two successive readings taken during the 90 minutes.

INTERPRETATION OF RESULTS

Having carried out the test on a group of three rabbits, repeat if necessary on further groups of three rabbits to a total of four groups.

Depending on the results obtained tabulate the results in the following manner.

NUMBER OF RABBITS	MATERIAL PASSED IF SUM OF RESPONSE DOES NOT EXCEED.	MATERIAL FAILED IF SUM OF RESPONSE EXCEEDS.
3 RABBITS	1.15C	2.65C
6 RABBITS	2.80C	4.30C
9 RABBITS	4.45C	5.95C
12 RABBITS	6.10C	7.60C