IP 2010 2.2.4.

DEPRESSOR SUBSTANCES

MethodE. End-Point Chromogenic Method Test Animal

Preparation of test solntions. Unless otherwise prescribed, Use a healthy, adult cat, either male or non-pregnant female,
prepare the solutions to be employed in the test using water weighing not less than 2 kg. Weigh the cat and anaesthetise it
BET. If necessary, adjust the pH of the solution under by intraperitoneal injection of an anaesthetic substance such
examination to 6.0 to 8.0 using sterile O.lM hydrochloric acid as chloralose or a suitable barbiturate that allows maintenance
BET, O.lM sodium hydroxide BET or a suitable buffer prepared of a uniform blood pressure. Immobilize the animal, protect it
with water BET. from loss of body heat and maintain it so that the rectal
temperature remains within physiological limits. Introduce a
Prepare the test solution at a suitable dilution. Prepare a
tube into the trachea. Expose a carotid or other suitable artery,
reagent blank and not less than three dilutions of CSE in water
separate it from surrounding tissues, insert a cannula fIlled
BET to prepare a linear standard curve. Use water BET as
with heparinised saline solution and connect to a device
negative control and one positive control. The positive control
capable of recording the blood pressure continuously. Then
consists of the test solution spiked with CSE to give an
expose a femoral vein and insert another cannula filled with
endotoxin concentration at the middle or below the middle
heparinised saline solution to facilitate intravenous injection
point of the standard curve (PPC).
of solutions of histamine and of the substance under
Method. Carry out the procedure described under Test for examination.
interfering factors. The chromogenic substrate and lysate are
Determine the sensitivity of the animal to histamine by injecting
added to the solution and incubated for the recommended
intravenously, at regular intervals, of not less than 5 minutes,
time. Stop the reaction and measure the absorbance at the
doses of standard histamine solution corresponding to 0.05,
wavelength specified by the lysate manufacturer.
0.1 and 0.15 /lg of histamine per kg of body weight of the
Perform the linear regression analysis of the absorbance on animal. Repeat the injection of the dose of 0.1 !J.g per kg at
the endotoxin concentration using standard statistical least three times. Administer the second and subsequent
methods (method ofleast squares is usually suitable). Do not injections not less than 1 minute after the blood pressure has
average the absorbance values of the r,eplicates of each returned to a constant level. Use the animal for the test only if
standard before performing the linear correlation regression the responses to the graded doses are clearly different and
analysis. Determine the endotoxin concentration of the test the responses to the repeated injeGtions of the dose of 0.1 /lg
solution from the standard curve. per kg are approximately the same and correspond to a decrease
Interpretation of results. The assay is valid only if in pressure of not less than 20 mmof mercury.
(a) the standard curve is linear for the range of CSE Method
concentrations used;
Dissolve the substance under examination in sufficient saline
(b) the co-efficient of correlation, r, is not less than 0.980;
solution or other diluent prescribed in the individual
(c) the mean percentage recovery of the added endotoxin monograph, to give the test solution of the concentration
in positive product control is between 50 per cent and specified in the monograph. Follow the same time schedule
150 per cent. established during the injection of standard histamine solution.
The product under examination meets the requirements of the Inject intravenously per kg of the cat's weight, 1.0 ml of
test if the mean endotoxin content of the replicates, after standard histamine solution followed by an injection of the
correction for dilution and concentration, is less than the specified amount of the test solution and fmally 1.0 ml of
endotoxin limit stated in the individual monograph. standard histamine solution. The second and third injections
are given not less than, 1 minute after the blood pressure has
returned to a constant level. When a common cannula is used
2.2.4. Depressor Substances for both the standard histamine solutions and test solutions,
each injection of the standard and the test solutions should
Special Reagents be immediately followed by an injection of approximately
2.0 ml of saline solution to flush any residues from the tubing.
Heparinised saline solution. A sterile saline solution
Measure the change in blood pressure following each of the
containing 50 Units of heparin in 1 ml.
three injections. The depressor response to the test solution
Standard histamine solution. Dissolve a suitable quantity of is not greater than one-half of the mean depressor response
histamine dihydrochloride or histamine acid phosphate in to the two associated doses of the standard histamine solution.
sufficient water or saline solution to produce a solution Ifthis requirement is not met, continue the series of injections
containing 0.1 mg ofhistamine, CsH9N3, per ml. Make suitable similarly until it consists of five doses, of which the three
dilutions with the solvent used for preparing the solution. doses of 1.0 ml each of standard histamine solution are

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2.2.5. TEST FOR COLONY~FORMING UNlTS (CFU) IP 2010

alternated with two doses of the test solution. Measure the Dipotassium hydrogen phosphate 0.5 g
change in blood pressure folloWing each of the. additional (KZHPq4)
injections.. The substance passes the test if the depressor Glycerin 60.0rnl
response to each dose of the test solution is not greater than.
Distilled water to 1000 ml
the mean of the respective depressor responses to the
associate doses of the standard histamine solution represen- Dissolve the solid ingredients in 50 rnl of distilled water by
ting 0.1 J.lg of histamine per kg. warming on a water-bath. Add glycerin and sufficient distilled
water to produce 1000.0 rnl , mix well and filter. Adjust the pH
If the depressor response to either dose of the test solution is
of thefiltrate to 7.2 ± 0.2 with 5 M sodium hydroxide. Sterilise
greater than the mean of the depressor response to the
by heating at 121 0 for 30 minutes. Store the medium in a light
associated doses of the standard histamine solution the test
resistant container in a cold place.
may be continued in the same animal or in another animal
similarly prepared and tested for responses to the standard b. Phosphate buffer solution
histamine solution. If the test continued in the same animal Dipotassium hydrogen phosphate 1.452 g
after the last dose of the standard histamine solution of the
Sodium dihydrogen phosphate 7.601 g
initial series, administer four more injections, of which two are
doses of the test solution and two are doses of 1.0 rnl each of Sodium chloride 4.8g
the standard histamine solution alternately in sequence. If the Distilled water to 1000 ml
test is continued in another animal, prepare a fresh solution of Dissolve the solids in sufficient distilled water to produce
the substance under examination from an independent 1000.0 rnl. Warm on a water-bath, if necessary, and filter.
container or containers of the substance and inject a series of
five doses comprising the standard histamine solution and c. Polysorbate 80 solution
test solution in accordance with the initial injection sequence. Polysorbate 80 10 ml
Measure the change in blood pressure following each of the
Phsophate buffer solution 90 ml
additional injections. Compute the difference between each
0
response to the dose of the test solution and the mean of the Mix and sterilise by heating at 121 for 20 minutes. Store in a
associated doses of the standard histamine solution in the cold place.
entire series, initial and additions, and calculate the average d. Dilute Sauton's solution
of all such differences is such that in the specified dose the
Sauton's fluid medium 1000 ml
depressor response to the test solution is not greater than the
depressor response to the dose of the standard histamine Distilled water to 3000ml
solution representing 0.1 J.lg of histamine per kg and if not Mix well and adjust the pH to 7.2 ± 0.2. Distribute into suitable
more than one-half of the depressor responses to the test containers. Sterilise by heating at 121 0 for 20 minutes.
solution are greater than the mean of the respective depressor
responses to the associated doses of the standard histamine Add 5 rnl of sterile polysorbate solution to 600 ml of dilue
solution representing 0.1 /lg of histamine per kg. sauton's solution immediately before use.
2. Lowenstein - Jensen Medium: LJ Medium

2.2.5 Test for Colony Forming Units (CFU) a. Mineral salt solution
Potassium hydrogen phosphate (K2HP04) 2.4 g
The number of Colony Forming Units (CFU) must be
determined on the contents of at least 5 containers of the Magnesium sulphate (MgS04) O.24g
freeze-dried vaccine. If the containers are vacuum sealed, check Magnesium citrate 0.6 g
for vacuum before use. L- Asparagine 3.6 g
Glycerin 12.0ml
Special reagents
Distilled water 600ml
1. Dilnte Santon's Medium
Dissolve the solid ingredients in 50 rnl of distilled water by
a. Sauton 's fluid medium warming on a water-bath. Add glycerin and 5 rnl of distilled
Ferric ammonium citrate (brown) 0.05 g water and mix well. Sterilise by heating at 121 0 for 25 minutes.
L-Asparagine 4.0 g b. Malachite green solution
Citric acid 2.0 g Prepare a 2 per cent w/v solution of malachite green in sterile
Magnesium sulphate (MgS04,7HzO) 0.5 g water with aseptic precautions, allowing the dye to dissolve

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