1.1 The tests for sterility are intended for detecting the presence of viable forms of bacteria, fungi and yeasts in or on pharmaceuticals preparations.
4.2.1 Dissolve the solids in the water, warming slightly to effect solution . Cool to room temperature and add, if necessary, sufficient 0.1N sodium Hydroxide to give a final pH after sterilization between 7.1 and 7.5. Filter, if necessary, to clarify, distribute into suitable containers and sterilize in an autoclave at 121 Deg. C. for about twenty minutes
4.3 INCUBATION TEMPERATURE::
4.3.1.Incubate portions of the media intended for the detection of bacteria at 30 Deg.C. to 35 Deg.C. and portions of those intended for the detection of fungi at 20Deg. to 25Deg.C.
4.4 NUTRITIVE PROPERTIES (For growth promotion and sterility validation test):
4.4.1. Incubate each medium to be used in the test with about 100 viable micro organisms.
i) FOR AEROBE: Staphylococcus aureus (NCTC 10788, ATCC 6538)
ii) FOR A SPORE FORMING AEROBE: Bacillus subtilis (ATCC 6633, NCI M 8054)
iii) FOR ANAEROBE: Clostridium sporogenes ( ATCC 11437; NCTC 532)
iv) FOR FUNGUS : Candida albicans(ATCC 10231 ; NCYC+ 854)
Aspergillus niger ATCC - 16404
4.5 EFFECTIVENESS OF MEDIA AND VALIDATION OF STERILITY TEST:
4.5.1 Prepare at least two separate containers of the medium for each, organism, aerobic , anaerobic and fungus and to each add the preparation being examined in the same quantity, and treated in the same manner, as in the test for sterility. Inoculate the separate containers with about 100 viable spores ( 0.1 ml of a suitably diluted suspension) of a suitable strain of an anaerobic organism, ( Clostridium sporogenes); aerobic organism (S. aureus) and for fungus (Candida albicans). Prepare a similar set of containers omitting the preparation being examined and inoculate them in the same manner. Incubate all the containers at 30 Deg.C.to 35 Deg.C. for aerobic and anaerobic bacteria and 20 to 22Deg.C. for fungi. If during the incubation the microbial growth is similar in the presence and the absence of the preparation being examined, the latter has no antimicrobial action under the conditions of the test and the test for sterility may be carried out without modification. If the cultures containing the preparation being examined show weaker growth, delayed growth or no growth of micro-organisms when compared with the controls, the preparation being examined has an antimicrobial activity which must be eliminated by filtration, dilution before or during the test for sterility. Establish that the antimicrobial properties of the preparation have been effectively removed by repeating the test for effectiveness of media.
5. STERILITY
The tests for sterility comprise the test for aerobic, anaerobic and for fungi.
5.1 GENERAL PROCEDURE:
5.1.1 Cleanse the exterior surface of ampoules, closures of vials and bottles with a suitable antimicrobial agent and gain access to the contents in a suitable aseptic manner. If the contents are packed in a container under vacuum, admit sterile air by means of a suitable sterile device, such as a needle with non absorbent cotton.
5.1.2 Select the number of samples to be tested from Table 1.
TABLE - 1
|
NUMBERS OF ITEMS IN THE BATCH
|
MINIMUM RECOMMENDED TO BE TESTED
|
|
1. INJECTABLE
PREPARATIONS
Not more than 100 containers
|
10% or 4 containers which
ever is the greater.
|
|
More than 100 but not more
than 500 containers
|
10 containers.
|
|
More than 500 containers
|
2% or 20 containers which
ever is less.
|
|
2. OPHTHALMIC AND OTHER NON
INJECTABLE
PREPARATIONS:
|
|
|
Not
more than 200 containers
|
5% or 2 containers
which ever is greater.
|
|
More than 200 containers
|
10 containers.
|
|
3. BULK SOLIDS
|
|
|
Less than 4 containers
|
Each container
|
|
4 Containers but not more than50 containers.
|
20% or 4 containers which
ever is the greater.
|
|
More than 500 containers.
|
2% or 10 containers which
ever is the greater
|
5.1.3 TWO METHODS ARE USED FOR STERILITY TEST:
5.1.3.1 MEMBRANE FILTRATION METHOD
5.1.3.1.1 APPARATUS: A suitable unit consists of a closed reservoir and a receptacle between which a properly supported membrane of appropriate 6 porosity is placed.
5.1.3.1.2 MEMBRANE: A Diameter is approximately 47 mm of 0.45 micron and a flow rate of 55 to 75 ml of water per minute at a pressure of 70 cm of mercury.
5.1.3.1.3 FLUID A (Diluting Fluid): Dissolve 1 gm of bacteriological peptone to make 1 litre, filter or centrifuge to clarify, adjust to pH 7.1 ±0.2 dispense into flask in 100 ml. quantities, and sterilize at 121 Deg.C. for 18-20 minutes.
5.1.3.1.4 FLUID B (Diluting Fluid): If the test sample contains lecithin or oil, use fluid A to each litre of which has been added 1 ml. of polysorbate 80, adjust to pH 7.1 u 0.2, dispense into flask and sterilise at 121 Deg. C for 18- 20 minutes.
NOTE: Any diluent that does not manifest antimicrobial activity and does not effect the porosity of the membrane may be suitable for dissolving a preparation under test for sterility.
5.1.3.1.5 QUANTITIES OF SAMPLE TO BE USED FOR EACH MEDIUM:
TABLE- A :
|
1) FOR INJECTABLE PREPARATRIONS:
|
|
Quantity in each container
|
Minimum quantity to be
used for each
culture medium.
|
|
FOR LIQUIDS:
|
|
|
i. Less than 1 ml.
|
The whole contents of a
container.
|
|
ii. 1 ml or more but less than 4 ml.
|
Half of the contents of a
container .
|
|
iii. 4 ml. or more but less
than 20 ml.
|
2 ml.
|
|
Iv. 20 ml. or more but less
than100 ml.
|
10 percent of the contents
of a container unless otherwise indi cated in
the monograph.
|
|
v. 10 ml. or more
|
Not less than half the
contents of the container unless otherwise specified.
|
|
FOR SOLIDS:
|
|
|
Less than 50 mg
|
The whole contents of a
container.
|
|
50 mg or more but less than
300 mg.
|
Half the contents of a
container but not less than 50 mg.
|
|
300 mg. to 5.0 gm.
|
150 mg.
|
|
Greater than 5.0 gm.
|
500 mg.
|
TABLE -B
|
2) FOR OPHTHALMIC AND OTHER NON INJECTABLE
PREPARATIONS:
|
|
TYPE OF PREPARATIONS
|
QUANTITY TO BE MIXED
|
QUANTITY TO BE USED FOR EACH CULTURE MEDIUM
|
|
Ophthalmic solutions and other non-injectable liquid
preparations.
|
10 to 100 ml.
|
5 to 10 ml.
|
|
Other preparations; preparations soluble in water or
appropriate solvents; insoluble preparations to be suspended or emulsified.
(Ointments and creams)
|
1 to 10 gm
|
0.5 to 1 gm.
|
5.1.3.1.6 METHOD OF TEST:
i) AQUEOUS SOLUTIONS:
Draw the liquid according the table rapidly through the filter with the aid of vacuum. If the solution has antimicrobial properties, wash the membrane not less three successive quantities, each of approximately 100 ml, of sterile fluid A. Filter aseptically cut the membrane in half, if only one is used put one half of the membrane, in 100ml, of soybean casein digest medium, and incubate at 20 Deg. C to 25 Deg. C for not less than 14ays. Similarly other half of the Membrane, in 100 ml of fluid thioglycollate medium, and incubate at 30 Deg. C to 35 Deg.C for not less than 14 days.
ii) LIQUIDS IMMISCIBLE WITH AQUEOUS VEHICLES AND SUSPENSIONS:
Method is same as aqueous but add a sufficient quantity of Fluid A to the pooled sample to achieve rapid filteration. Sterile enzyme preparations such as penicillinase or cellulase may be added to Fluid A to aid in dissolving insoluble substances. If the sample contains lecithin use fluid B for diluting.
iii) OILS AND OILY SOLUTIONS:
Filter oils or oily solutions of sufficiently low viscosity without dilution through a dry membrane. Dilute viscous oils as necessary with a suitable sterile diluent such as isopropyl myristate, that has been shown not to have antimicrobial properties under the conditions of the test. Allow the oil to penetrate the membrane and filter, applying pressure or suction gradualy. Wash the membrane by at least three successive quantities of approximately 100 ml. of sterile fluid B. Further proceed as described in aqueous solution.
IV. OINTMENTS AND CREAMS:
Dilute ointments in a fatty base and emulsion of the water in oil type, to give a fluid concentration of 1% w/v, by heating, if necessary, to not more than 40 Deg.C with a suitable sterile diluent that does not have antimicrobial properties. Further proceed as described in oily solutions.
NOTE: for ointments and oils that are not soluble in isopropyl myristate, use method B
V. SOLUBLE SOLIDS:
Collect the sample according to table and proceed same as described in aqueous solutions and proceed same as in aqueous solutions.
VI. STERILE DEVICES:
Aseptically pass a sufficient volume of fluid B through each of not less than twenty devices so that not less than 100 ml is recovered from each device. Collect the fluids in a sterile containers, and filter the entire volume collected through membrane filter funnel and further proceed as in aqueous solutions.
5.1.4 METHOD-B DIRECT INOCULATION:
5.1.4.1 Sample for inoculation is collected according to table c
TABLE C
|
CONTAINER CONTENT
|
MINIMUM QUANTIY OF PRODUCT
|
MINIMUM VOLUME OF CULTURE MEDIUM(ML)
|
|
FOR LIQUIDS:
|
|
Less than 1 ml.
|
Total content
|
15
|
|
1ml or more but less than 5 ml.
|
Half the contents
|
20
|
|
5 ml. or more but less than 20 ml.
|
2 ml.
|
20
|
|
20 ml. or more but less
than 50 ml.
|
5 ml.
|
40
|
|
50 ml. or more but less
than 100 ml.
|
10 ml
|
80
|
|
FOR SOLIDS
|
|
Less than 50 mg.
|
Total contents
|
40
|
|
50 mg or more but less than
200 mg.
|
Half the contents
|
80
|
|
200 mg. or more
|
100 mg.
|
80
|
5.1.2.1 METHOD OF TEST:
i) AQUEOUS SOLVENT AND SUSPENSIONS:
Remove the liquid from test containers with a sterile pipette or syringe and needle. Aseptically transfer the volume according to table C from each containers to a vessel of culture medium. Mix the liquid with the medium, but do not aerate excessively, Incubate in the specified media for not less than fourteen days.
When the material being tested renders the medium turbid, so that the presence or absence ofmicrobial growth cannot be determined readily by visual examination, transfer suitable portions of the medium to fresh vessels of the same medium between the third and seventh days after the test is started. Continue incubation of the original and of the transfer vessels for not less than a seven additional days after the transfer and for a total of not less than fourteen days.
ii) OILS AND OILY SOLUTIONS:
Use media to which have been added 1% w/v polysorbate 80 or other suitable emualsifying agents, in an appropriate concentration, shown not to have any antimicrobial properties, carry out the test as described aqueous solutions and suspensions.
iii) OINTMENTS
Prepare by diluting ten fold in a suitable sterile diluent such as Fluid B or any other aqueousvehicle. Capable of dispersing the test material homogenously throughout the fluid mixture. Mix 10 ml. of the fluid mixture so obtained with 80 ml. of medium and proceed as directed under aqueous solutions and suspensions.
iv.SOLIDS:
Transfer the quantity of the preparation to be examined to the quantity of medium specified in Table C and mix, the conditions of incubation being the same as for aqueous solution and suspensions.
v.STERILE DEVICES.
For articles of such size and shape as to permite complete immersion in not more than 1000 ml of culture medium, test the intact article, using the appropriate media and incubate as directed under general procedure.
5.1.5. GENERAL PROCEDURE:
For transfusion assemblies or where the size of an item makes immersion impracticable and only the liquid path way must be sterile, flush the lumen of each of twenty units with a sufficient quantity of fluid thiolycollate medium and the lumen of each of twenty units with a sufficient quantity of soybean- casein digest medium to yield a recovery of not less than 15 ml. of each medium, and incubate with not less than 100 ml. of each of the two media as directed
5.1.5.1 OBSERVATION AND INTERPRETATION OF RESULTS:
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 can be 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 is invalid and may therefore be recommended, perform a retest using the same number of samples, volumes to be tested and the media as in the original test. If no evidance of microbial growth is then found, passes the test for sterility. If evidence of microbial growth is found, isolated and identify the organism. If they are not readily distinguishable from those growing in the containers reserved in the first test the preparation being examined fails the test for sterility. If they are readily distinguishable from those growing in the containers reserved in the first test, perform a second re-test using twice the number of samples. If no evidence of microbial growth is found in the second retest, passes the test for sterility. If evidence of growth of any micro-organisms is found in the second re-test the preparation being examined fails the test for sterility.
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