1.0 OBJECTIVE
The test for Anti-microbial preservative - Effectiveness is provided to determine compliance with the requirements given in individual monograph / specifications.
2.0 PRINCIPLE
Preservative efficacy test is basically a challenge test in which the function of a preservative in pharmaceutical preparation use to be checked by challenging it with different microorganisms (bacteria, fungi and yeast.)
Test Organisms
Aspergillus niger IMI 147 007 (ATCC 16404, IP 1431.83)
Candida albicans NCPE 3179 (ATCC 10231, IP 48.72)
Pseudomonas aeruginosa NCIMB 8626 (ATCC 9027, CIP 82.118)
Staphylococcus aureus NCTC 10788 (NCIMB 9518, ATCC 6538, CIP 4.13) .
Single-strain challenges are used and the designated micro-organisms are supplemented by other strains or species that may represent likely contaminants to the preparation. For example, Escherichia coli [NCIMB 8545 (ATCC 8739, CIP 53.126)] is used for all oral preparations and Zygosaccharomyces Rouxii[NCYC 381 (IP 2021.92)] for oral preparations containing a high concentration a sugar.
Medium
a. Soyabean Casein Digest Agar Medium . (or Tryptone Soya Agar)
Pancreatic digest of Casein 17.0 g
Papaic digest of Soyabean meal 3.0 g
Sodium Chloride 5.0 g
Dibasic Potassium Phosphate 2.5 g
Dextrose (C6H12O.H2O) 2.5 g
Distilled water 1000 mL
Final pH after Sterilization 7.3 ± 0.2
Dissolve the solids in the water, warming slightly to effect solution. Cool to room temperature and add, if necessary, sufficient 0.1 N Sodium Hydroxide to give a final pH after sterilization of between 7.1 and 7.5. Filter, if necessary, to clarify, distribute into suitable containers and sterilize in an autoclave at 121oC for about 15 min.
b. Sabouraud Agar (Sabouraud Glucose Agar)
D- Glucose monohydrate 4.0 g
Peptones (meat and casein) 10.0 g
Agar 15.0 g
Water 1000 mL
Dissolve the ingredients in the water, adjust to give an expected pH of 5.4 to 5.8 and sterilize by heating in an autoclave at 121oC for 15 min.
All the above media should be incubated for 24 h at 37oC before use. Any contaminated media should be discarded.
Instead of preparing media, one can also use dehydrated media of Hi media/Difco and rehydrate the required quantify as per instructions on the bottle label, dispense in required quantities and sterilize at 15 psi at 121oC for 15 min.
Preparation of Inoculum
1. Preparatory to the test, inoculate the surface of a Tryptone soya agar plate for bacteria or sabouraud agar plate for fungi, with the recently grown stock culture of each of the specified micro-organisms.
2. Incubate the bacterial cultures at 30oC - 35oC for 18 - 24 h, the culture of Candida albicans at 20oC to 25oC for 48 h, and culture of Aspergillus niger at 20oC to 25oC for 7 days or until good sporulation is obtained.
3. Sub-cultures may be needed after revival before the organism is in its optimal state, but it is recommended that the number of subcultures be kept to a minimum.
4. To harvest the bacterial and Candida albicans cultures, use a sterile suspending fluid containing 0.9% w/v of Sodium Chloride and 0.1%w/v of Peptone for dispersal and transfer of the surface growth into a suitable vessel.
5. Add sufficient suspending fluid to reduce microbial count to about 108 micro-organisms per mL.
6. To harvest the Aspergillus niger culture, use a sterile suspending fluid containing 0.9% w/v of Sodium Chloride and 0.05% w/v of Plysorbate 80 and adjust the spore count to abut 108 per mL by adding the same solution.
7. Remove immediately a suitable sample from each suspension and determine the number of colony-forming units per mL in each suspension by plate count or by membrane filtration.
8. The value serves to determine the inoculum and the base line to use in the test.
9. The suspensions shall be used immediately.
As per IP
Procedure
1. Inoculate each original product container or product tube (when original container is not suitable for inoculation with sterile syringe fitted with needle, transfer 20 mL per capped bacteriological tube) with one of the standardized microbial suspension using a ration equivalent to 0.1 mL of inoculum suspension to 20 mL of product, and mix.
2. Final concentration should be between 1 x 105 and 1 x 106 micro-organisms per mL of product.
3. Determine, the number of viable micro-organisms by plate count method in each inoculum suspension and from there calculate the initial concentration of micro-organisms per mL of product under test.
4. Incubate the inoculated containers or tubes at 20o C to 25o C.
5. Determine the viable count (by plate count method at 7,14,21 and 28 days subsequent to inoculation).
6. Record any change, if observed in appearance.
As per BP
Procedure
1. To count the viable micro-organisms in the inoculated products, use the Agar medium
2. Inoculate a series of containers of the product to be examined, each with a suspension of one of the test organisms to give an inoculum of 105 to 106 micro-organisms per g or per mL of the preparation.
3. The volume of the suspension of inoculum does not exceed 1 % of the volume of the product.
4. Mix thoroughly to ensure homogeneous distribution.
5. Maintain the inoculated containers at 20 oC to 25 oC, protected from light.
6. Remove a suitable sample from each container, typically 1 mL of 1 g quantities, at zero hour and at appropriate intervals according to the type of the product and determine the number of viable microorganisms by plate count or membrane filtration.
7. Ensure that any residual anti-microbial activity of the product is eliminated by dilution, by filtration or by the use of a specific incubator.
8. When dilution procedures are used, due allowance is made for the reduced sensitivity in the recovery of small numbers of viable micro-organisms.
9. When the specific inactivator is used, the ability of the system to support the growth of the test organisms in confirmed by the use of appropriate controls. The procedure is validated to verify its ability to demonstrate the required reduction in count of viable micro-organisms.
As per USP
Procedure
1. Where the product container can be entered aseptically, such as with a needle and syringe through a rubber stopper, conduct the test in 5 original product containers.
2. If the product container is such that it can not be entered aseptically, transfer 20 mL samples of the product to each of five sterile, capped bacteriological tubes of suitable size.
3. Inoculate each tube or product container with one of the standardized microbial suspensions, using a ratio equivalent to 0.10 mL of INOCULUM to 20 mL of product, and mix.
4. A suitable concentration of test micro-organisms should be added so that the concentration in the test preparation immediately after inoculation is between 100,000 and 1,000,000 micro-organisms per mL.
5. Determine the number of viable micro-organisms in each inoculum suspension, and calculate the initial concentration of micro-organisms per mL of product under test by the plate-count method.
6. Incubate the inoculated containers or tubes at 20 oC to 25 oC.
7. Examine the containers or tubes at 7, 14, 21, and 28 days subsequent to inoculation.
8. Record any changes observed in appearance, and determine by the plate-count procedure the number of viable micro-organisms present at each of these time intervals.
9. Using the theoretical concentrations of micro-organisms present at the start of the test, calculate the percentage change in the concentration of each micro-organisms during the test.
INTERPRETATION:
The preservative is effective in the product examined if:
1. The concentration of viable bacteria are not more than 0.1% of the initial concentrations by the fourteenth day.
2. The concentrations of viable yeasts and molds remain at or below the initial concentrations during the first 14 days.
3. The concentration of each test micro-organism remains at or below these designated levels during the remainder of 28 day test period.
The criteria for evaluation of anti-microbial activity are given in the appropriate table below in terms of the log reduction in the number of viable micro-organisms using as baseline the value obtained for the inoculum.
The test for Anti-microbial preservative - Effectiveness is provided to determine compliance with the requirements given in individual monograph / specifications.
2.0 PRINCIPLE
Preservative efficacy test is basically a challenge test in which the function of a preservative in pharmaceutical preparation use to be checked by challenging it with different microorganisms (bacteria, fungi and yeast.)
Test Organisms
Aspergillus niger IMI 147 007 (ATCC 16404, IP 1431.83)
Candida albicans NCPE 3179 (ATCC 10231, IP 48.72)
Pseudomonas aeruginosa NCIMB 8626 (ATCC 9027, CIP 82.118)
Staphylococcus aureus NCTC 10788 (NCIMB 9518, ATCC 6538, CIP 4.13) .
Single-strain challenges are used and the designated micro-organisms are supplemented by other strains or species that may represent likely contaminants to the preparation. For example, Escherichia coli [NCIMB 8545 (ATCC 8739, CIP 53.126)] is used for all oral preparations and Zygosaccharomyces Rouxii[NCYC 381 (IP 2021.92)] for oral preparations containing a high concentration a sugar.
Medium
a. Soyabean Casein Digest Agar Medium . (or Tryptone Soya Agar)
Pancreatic digest of Casein 17.0 g
Papaic digest of Soyabean meal 3.0 g
Sodium Chloride 5.0 g
Dibasic Potassium Phosphate 2.5 g
Dextrose (C6H12O.H2O) 2.5 g
Distilled water 1000 mL
Final pH after Sterilization 7.3 ± 0.2
Dissolve the solids in the water, warming slightly to effect solution. Cool to room temperature and add, if necessary, sufficient 0.1 N Sodium Hydroxide to give a final pH after sterilization of between 7.1 and 7.5. Filter, if necessary, to clarify, distribute into suitable containers and sterilize in an autoclave at 121oC for about 15 min.
b. Sabouraud Agar (Sabouraud Glucose Agar)
D- Glucose monohydrate 4.0 g
Peptones (meat and casein) 10.0 g
Agar 15.0 g
Water 1000 mL
Dissolve the ingredients in the water, adjust to give an expected pH of 5.4 to 5.8 and sterilize by heating in an autoclave at 121oC for 15 min.
All the above media should be incubated for 24 h at 37oC before use. Any contaminated media should be discarded.
Instead of preparing media, one can also use dehydrated media of Hi media/Difco and rehydrate the required quantify as per instructions on the bottle label, dispense in required quantities and sterilize at 15 psi at 121oC for 15 min.
Preparation of Inoculum
1. Preparatory to the test, inoculate the surface of a Tryptone soya agar plate for bacteria or sabouraud agar plate for fungi, with the recently grown stock culture of each of the specified micro-organisms.
2. Incubate the bacterial cultures at 30oC - 35oC for 18 - 24 h, the culture of Candida albicans at 20oC to 25oC for 48 h, and culture of Aspergillus niger at 20oC to 25oC for 7 days or until good sporulation is obtained.
3. Sub-cultures may be needed after revival before the organism is in its optimal state, but it is recommended that the number of subcultures be kept to a minimum.
4. To harvest the bacterial and Candida albicans cultures, use a sterile suspending fluid containing 0.9% w/v of Sodium Chloride and 0.1%w/v of Peptone for dispersal and transfer of the surface growth into a suitable vessel.
5. Add sufficient suspending fluid to reduce microbial count to about 108 micro-organisms per mL.
6. To harvest the Aspergillus niger culture, use a sterile suspending fluid containing 0.9% w/v of Sodium Chloride and 0.05% w/v of Plysorbate 80 and adjust the spore count to abut 108 per mL by adding the same solution.
7. Remove immediately a suitable sample from each suspension and determine the number of colony-forming units per mL in each suspension by plate count or by membrane filtration.
8. The value serves to determine the inoculum and the base line to use in the test.
9. The suspensions shall be used immediately.
As per IP
Procedure
1. Inoculate each original product container or product tube (when original container is not suitable for inoculation with sterile syringe fitted with needle, transfer 20 mL per capped bacteriological tube) with one of the standardized microbial suspension using a ration equivalent to 0.1 mL of inoculum suspension to 20 mL of product, and mix.
2. Final concentration should be between 1 x 105 and 1 x 106 micro-organisms per mL of product.
3. Determine, the number of viable micro-organisms by plate count method in each inoculum suspension and from there calculate the initial concentration of micro-organisms per mL of product under test.
4. Incubate the inoculated containers or tubes at 20o C to 25o C.
5. Determine the viable count (by plate count method at 7,14,21 and 28 days subsequent to inoculation).
6. Record any change, if observed in appearance.
As per BP
Procedure
1. To count the viable micro-organisms in the inoculated products, use the Agar medium
2. Inoculate a series of containers of the product to be examined, each with a suspension of one of the test organisms to give an inoculum of 105 to 106 micro-organisms per g or per mL of the preparation.
3. The volume of the suspension of inoculum does not exceed 1 % of the volume of the product.
4. Mix thoroughly to ensure homogeneous distribution.
5. Maintain the inoculated containers at 20 oC to 25 oC, protected from light.
6. Remove a suitable sample from each container, typically 1 mL of 1 g quantities, at zero hour and at appropriate intervals according to the type of the product and determine the number of viable microorganisms by plate count or membrane filtration.
7. Ensure that any residual anti-microbial activity of the product is eliminated by dilution, by filtration or by the use of a specific incubator.
8. When dilution procedures are used, due allowance is made for the reduced sensitivity in the recovery of small numbers of viable micro-organisms.
9. When the specific inactivator is used, the ability of the system to support the growth of the test organisms in confirmed by the use of appropriate controls. The procedure is validated to verify its ability to demonstrate the required reduction in count of viable micro-organisms.
As per USP
Procedure
1. Where the product container can be entered aseptically, such as with a needle and syringe through a rubber stopper, conduct the test in 5 original product containers.
2. If the product container is such that it can not be entered aseptically, transfer 20 mL samples of the product to each of five sterile, capped bacteriological tubes of suitable size.
3. Inoculate each tube or product container with one of the standardized microbial suspensions, using a ratio equivalent to 0.10 mL of INOCULUM to 20 mL of product, and mix.
4. A suitable concentration of test micro-organisms should be added so that the concentration in the test preparation immediately after inoculation is between 100,000 and 1,000,000 micro-organisms per mL.
5. Determine the number of viable micro-organisms in each inoculum suspension, and calculate the initial concentration of micro-organisms per mL of product under test by the plate-count method.
6. Incubate the inoculated containers or tubes at 20 oC to 25 oC.
7. Examine the containers or tubes at 7, 14, 21, and 28 days subsequent to inoculation.
8. Record any changes observed in appearance, and determine by the plate-count procedure the number of viable micro-organisms present at each of these time intervals.
9. Using the theoretical concentrations of micro-organisms present at the start of the test, calculate the percentage change in the concentration of each micro-organisms during the test.
INTERPRETATION:
The preservative is effective in the product examined if:
1. The concentration of viable bacteria are not more than 0.1% of the initial concentrations by the fourteenth day.
2. The concentrations of viable yeasts and molds remain at or below the initial concentrations during the first 14 days.
3. The concentration of each test micro-organism remains at or below these designated levels during the remainder of 28 day test period.
The criteria for evaluation of anti-microbial activity are given in the appropriate table below in terms of the log reduction in the number of viable micro-organisms using as baseline the value obtained for the inoculum.
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