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Injection should be sterile, isotonic and free from foreign particles, such as
dust, fibers etc.They should be introduce through the same route for which they
are intended.
1. Sterility test
2. Pyrogen test
3. Clarity test
4. Leakage test
5. Assay
STERILITY TEST
The test for sterility are intended for detecting the presence of viable forms
of micro-organisms in or on pharmacopoeial preparations. The test must be
carried out under conditions designed to avoid accidental contamination of the
product during the test. Precautions taken for this purpose should not adversely
affect any micro-organisms which should be revealed in the test.
The tests for sterility are designed to reveal the presence of micro-
organisms in the samples used in the tests; interpretation of results is based on
the assumption that the contents of every container in the batch, had they been
tested, would also have complied with the tests. Since every container cannot be
tested, a sufficient number of containers should be examined to give a suitable
degree of confidence in the results of the tests.
CULTURE MEDIA
For use with clear fluid products. Mix the ingredients other than the
thioglycollate and the resazurin, in the order given above in a mortar, with
through grinding. Stir in some heated distilled water, transfer to a suitable
container, add the remainder of the water and complete the solution by heating in
a boiling water-bath. Add the Sodium thioglycollate, then 1M Sodium hydroxide, if
necessary, so that (after sterilisation) the medium will have a pH of 7.1 + 0.2.
Reheat the solution, but do not boil, filter (if necessary) through a moistened filter
paper and add the resazurin solution.
Dissolve the solids in distilled water, warming slightly to effect solution. Cool to
room temperature and add, if necessary, sufficient 0.1M sodium hydroxide to give
a final pH of 7.1 + 0.2 after sterilisation. Filter, if necessary, distribute into suitable
containers and sterilise in an autoclave at 121º for 20 minutes.
NOTE – Where fluid thioglycollate medium and soyabean- casein digest medium
are to be used in Method B for samples of the penicillin or cephalosporin class of
antibiotic, eseptically transfer to each tube of medium a quantity of penicillinase
sufficient to inactivate the amount of antibiotic in the sample under test.
Determine the appropriate quantity of penicillinase to be used for this purpose by
using a penicillinase preparation that has been assayed previously for its
penicillin or cephalosporin-inactivating power. Alternatively, confirm that the
appropriate quantity of penicillinase has been transferred to a tube of fluid
thioglycollate medium by adding to it an amount of penicillin or cephalosporin
antibiotic equivalent to the amount of antibiotic in the sample being examined,
inoculating the medium with 1 ml of a 1:1000 dilution of an 18- to 24 hour culture
of Staphylococcus aureus (ATCC 29737) in fluid thioglycollate mediaum and
incubating it for 24 hour at 30º to 35º at this time typical microbial growth must be
observed. Perform this confirmatory test in an area completely separate from that
used for sterility testing.
Precautions : The tests for sterility should be carried out under conditions
designed to avoid accidental contamination of the product during the test using,
for example, a laminar sterile airflow cabinet. The precautions taken to avoid
contamination must be such that they do not affect any micro-organisms that
should be revealed in the test.
At intervals during the incubation period, and at its conclusion, examine the
media for macroscopic evidence of microbial growth. If no evidence of growth is
found, the preparation being examined passes the test for sterility. If evidence of
microbial growth is found, reserve the containers showing this and, unless it is
demonstrated by any other means that their presence is due to causes unrelated
to the preparation being examined and hence that the tests for sterility are invalid
and may therefore be recommenced, perform a retest using the same number of
samples, volumes to be tested and the media as in the original test. If no
evidence of microbial growth is then found, the preparation being examined
passes the tests for sterility. If evidence of microbial growth is found, isolate and
identify the organisms. If they are readily distinguishable from those growing in
the containers reserved in the first test, perform a second retest using twice the
number of samples. If no evidence of microbial growth is found in the second
retest, the preparation being examined passes the tests for sterility. If evidence of
growth of any micro-organisms is found in the second retest, the preparation
being examined fails the tests for sterility
PYROGEN TEST
The test involves measurement of the rise in body temperature of rabbits
following the intravenous injection of a sterile solution of the substance being
examined. It is designed for products that can be tolerated by the test rabbit in a
dose not exceeding 10ml per kg injected intravenously within a period of not
more than 10 minutes.
Test Animals
Use healthy, adult rabbits of either sex, preferably of the same variety,
weighing not less than 1.5 kg, fed on a complete and balanced diet and not
showing loss of body weight during the week preceding the test. House the
animals individually in an area of uniform temperature (± 2° ), preferably with
uniform humidity, and free from disturbances likely to excite them. Do not use
animals for pyrogen tests more frequently than once every 48 hours. After a
pyrogen test in the course of which a rabbits temperature has risen by 0.6° or
more, or after a rabbit has been given a test substance that was adjudged
pyrogenic, at least 2 weeks must be allowed to elapse the animals is used again.
Materials
All glassware, syringes and needles must be thoroughly washed with water
for injection and heated in a hot air oven at 250° for 30 minutes or at 200° for 1
hour. Treat all diluents and solutions for washing and rinsing of devices in a
manner that will assure that will assure that they are sterile and pyrogen-free.
Recording of Temperature
Use an accurate temperature-sensing device such as a clinical thermometer
or thermistor or other suitable probes that have been calibrated to assure an
accuracy of 0.1° and have been tested to determine that a maximum reading is
reached in less than 5 minutes. Insert the thermometer or temperature sensing
probe into the rectum of the test rabbit to a depth of about 5-cm. The depth of
insertion is constant for any one rabbit in any one test. If an electrical device is
used, it should be inserted in the rectum of the rabbit 90 minutes before the
injection of the solution being examined and left in position throughout the test.
After a period of time not less than that previously determined as sufficient,
record the rabbit's body temperature.
Preliminary Test (Sham Test)
If animals are used for the first time in a pyrogen test or have not been
used during the 2 previous weeks, condition them 1 to 3 days before testing the
substance being examined by injecting intravenously into them 10 ml per kg of
body weight of a pyrogen-free saline solution warned to about 38.5°. Record the
temperatures of the animals, beginning at least 90 minutes before injection and
continuing for 3 hours after injection of the solution being examined. Any animal
showing a temperature variation of 0.6° or more must not be used in the main
test.
Main Test
Carry out the test using a group of three rabbits.
Procedure:
Record the temperature of each animal at intervals of not more than 30
minutes, beginning at least 90 minutes before the injection of the solution being
examined and continuing for 3 hours after the injection. Not more than 40
minutes immediately preceding the injection of the test dose, record the "initial
temperature " of each rabbit, which is the mean of two temperatures recorded for
that rabbit at an interval of 30 minutes in the 40 minute period. Rabbits showing a
temperature variation greater than 0.2° between two successive readings in the
determination of "initial temperature " should not be used for the test. In any one
group of test animals, use only those animals whose " initial temperatures" do not
vary by more than 10 from each other ,and do not use any rabbit having a
temperature higher than 39.8° and lower than 38° Inject the solution being
examined slowly into the marginal vein of the ear of each rabbit over a period not
exceeding 4 minutes, unless otherwise prescribed in the monograph. The
amount of sample to be injected varies according to the preparation being
examined and is prescribed in the individual monograph. The volume of injection
is not less than 0.5 ml per kg and not more than 10 ml per kg of body weight.
Records the temperature of each animal at half-hourly intervals for 3 hours after
the injection. The difference between the "initial temperature " and the "maximum
temperature" which is the highest temperature recorded for a rabbit is taken to be
its response. When this difference is negative, the results is counted as a zero
response.Interpretation of results: if the sum of the responses of the group of
three rabbits is less than 0.6° , the preparation being examined passes the test.
If the response of any rabbit is 0.6° or more, or if the sum of the response of the
three rabbits exceeds 1.4° , continue the test using five other rabbits. If not more
than three of the eight rabbits show individual responses of 0.6° or more, and if
the sum of responses of the group of eight rabbits does not exceed 3.7° , the
preparation being examined passes the test.
CLARITY TEST
This limit test is suitable for revealing the presence of particles whose
longest axis, or effective linear dimension, is 10 µm or more. Alternative methods
such as the automatic measurement of particle size operating on the electrical
zone sensing principle or on the basis of light blockage may be employed
provided the results obtained are of equivalent reliability. However, where a
difference appears, or in the event of dispute, only the result obtained by the
procedure given in this Pharmacopoeia is conclusive.
Method:
10 50
25 5
50 Nil
LEAKAGE TEST
Definition
Leakage occurs when a discontinuity exists in the wall of a package that
can allow the passage of gas under the action of a pressure or concentration
differential existing across the wall. Leakage is mathematically defined as the
rate at which a unit of gas mass (or volume) .goes into or out of the leak under
specific conditions of temperature and pressure.
A method of testing the integrity of ampules containing a liquid therein for
leakage comprising the steps of:
(a) Immersing the ampules in an aqueous solution of a water soluble salt of a
dye,
(b) Applying negative pressure or a vacuum to the top of the solution containing
the dye to generate a pressure differential between said solution and the liquid
within the ampules at least once, and releasing said pressure or vacuum,
(c) Removing the ampules from the solution or draining the solution from the
container,
(d) Decontaminating the ampules by removing any solution containing dye
adhering to the external surface thereof.
(e) Color testing the ampules to detect faulty ampules into which dye has leaked,
(f) Rejecting said faulty ampules, if any, and subsequently.
ASSAY
Assay is performed according to the method given in the monograph of
that parenteral preparation in the pharmacopoeia. Assay is done to check the
quantity of medicament present in the parenteral preparation
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