NOTE-The test for the effectiveness of antimicrobial preservatives shall be demonstrated during development of pharmaceutical preparation and during the commercial manufacturing. The test is not intended to be used for routine control purpose.
The efficacy of antimicrobial preservation of a pharmaceutical preparation on its own or, if necessary, with the addition of a suitable preservative has to be ascertained during the development of the product. The primary purpose of adding antimicrobial preservatives to dosage forms is to prevent adverse effects arising from contamination by microorganisms that may be introduced inadvertently during or subsequent to the manufacturing process.
The efficacy of antimicrobial preservation of a pharmaceutical preparation on its own or, if necessary, with the addition of a suitable preservative has to be ascertained during the development of the product. The primary purpose of adding antimicrobial preservatives to dosage forms is to prevent adverse effects arising from contamination by microorganisms that may be introduced inadvertently during or subsequent to the manufacturing process.
However, antimicrobial agents should not be used solely to reduce the viable microbial count as a substitute for good manufacturing procedures. There may be situations where a preservative system may have to be used to minimize proliferation of microorganisms in preparations that are not required to be sterile. It should be recognized that the presence of dead microorganisms or their metabolic by-products may cause adverse reactions in sensitized persons.
Antimicrobial efficacy test must be demonstrated for multiple dose parenteral, otic, nasal, opthalmic, oral and topical products made with aqueous bases or vehicles, the effectiveness of any added preservatives, during the shelf-live of the preparations to ensure that the antimicrobial activity has not been impaired by storage. The tests apply only to the product in the original, unopened container in which it was supplied by the manufacturer.
The test consists of challenging the preparation in its final container with a prescribed inoculum of suitable microorganisms, storing the inoculated product at a prescribed temperature, withdrawing samples from the container at specified intervals of time and counting the organisms in the samples removed. The preservative properties of the product are considered adequate if, in the conditions of the test, there is a significant fall or no increase in the number of microorganisms in the inoculated preparation after storage for the times and at the temperatures prescribed.
The organisms specified for use in the tests are intended to be representative of those that might be expected to be found in the environment in which the preparation is manufactured, stored and used. However, they should be supplemented by other strains or species, especially those likely to be found in the conditions under a particular product is made or used, or that might offer a particular challenge to the type of product being tested. Single strain challenges (rather than mixed cultures) should be used throughout.
Candida albicans, ATCC 10231 *
Aspergillus niger ATCC 16404
Escherichia coli ATCC 8739
Pseudomonas aeruginosa ATCC 9027
Staphylococus aureus ATCC 6538
*Instead of Candida albicans, Zygosccharomyces rouxii. NCYC 381;
In order to prevent any phenotypic changes in the strains used, the organisms used in the test should not be more than 5 passages made from the original culture. One passage is defined as inoculation and growth of the organisms from existing culture to a fresh medium.
NOTE- All the media used in the tests should be tested for growth promotion.
The initial concentration of the viable organisms in each test preparation is estimated based on the concentration of the microorganisms in each of the standardized inoculum as determined by the pour plate method or membrane filtration method. Incubate the inoculated containers at room temperature.Determine the viable count by plate-count method at 7, 14, and 28 days subsequent to the inoculation. Record any changes observed in the appearance at these intervals. From the calculated concentration of cfu per ml present at the start of the test, calculate the percentage of reduction in cfu per ml for each organism at the stated test intervals and express the changes in terms of percentage of initial concentration.
i) For parenteral, ophthalmic, sterile nasal and optic preparations:
(a) the concentration of the viable bacteria are not more than 10 percent of the initial concentration at 7 days and not more than 0.1 percent of the initial concentration at 14 days and there is a further decrease in count at 28 days.
(b) there is no increase in yeast and mold count at 7, 14 and 28 days from the initial count.
ii) For topical preparations made with aqueous base, nonsterile nasal preparation and emulsions including those applied to mucous membrane: (a) the concentration of the viable bacteria are not more than 1 percent of the initial concentration at 14 days and there is a further decrease in count at 28 days. (b) there is no increase in yeast and mold count at 14 and 28 days from the initial count.
iii) For oral preparations: (a) the concentration of the viable bacteria are not more than 10 percent of the initial concentration at 14 days and there is a further decrease in count at 28 days. (b) there is no increase in yeast and mold count at 14 and 28 days from the initial count.
Antimicrobial efficacy test must be demonstrated for multiple dose parenteral, otic, nasal, opthalmic, oral and topical products made with aqueous bases or vehicles, the effectiveness of any added preservatives, during the shelf-live of the preparations to ensure that the antimicrobial activity has not been impaired by storage. The tests apply only to the product in the original, unopened container in which it was supplied by the manufacturer.
The test consists of challenging the preparation in its final container with a prescribed inoculum of suitable microorganisms, storing the inoculated product at a prescribed temperature, withdrawing samples from the container at specified intervals of time and counting the organisms in the samples removed. The preservative properties of the product are considered adequate if, in the conditions of the test, there is a significant fall or no increase in the number of microorganisms in the inoculated preparation after storage for the times and at the temperatures prescribed.
The organisms specified for use in the tests are intended to be representative of those that might be expected to be found in the environment in which the preparation is manufactured, stored and used. However, they should be supplemented by other strains or species, especially those likely to be found in the conditions under a particular product is made or used, or that might offer a particular challenge to the type of product being tested. Single strain challenges (rather than mixed cultures) should be used throughout.
Precautions
Challenge tests should be conducted under conditions that prevent accidental contamination of the product during the test but the precautions taken to prevent contamination should not affect the survival of organisms in the product being examined.Test organisms:
The following test organisms are used in the test.Candida albicans, ATCC 10231 *
Aspergillus niger ATCC 16404
Escherichia coli ATCC 8739
Pseudomonas aeruginosa ATCC 9027
Staphylococus aureus ATCC 6538
*Instead of Candida albicans, Zygosccharomyces rouxii. NCYC 381;
In order to prevent any phenotypic changes in the strains used, the organisms used in the test should not be more than 5 passages made from the original culture. One passage is defined as inoculation and growth of the organisms from existing culture to a fresh medium.
NOTE- All the media used in the tests should be tested for growth promotion.
Preparation of inoculum:
Grow each of the bacterial species separately in Casein soyabean digest agar and incubate them at 30° to 35° for 8 to 24 hours. Grow Candida albicans on Sabouraud dextrose agar and incubate at 20° to 25° for at least 48 hours. Grow Aspergillus niger on Sabouraud dextrose agar at 20° to 25° for 5-7 days. After incubation, harvest the growth and resuspend each of the organisms separately in sterile to obtain a microbial count of lx108 cfu per ml. To suspend spores of Aspergillus niger 0.05per cent polysorbate 80 may be added to the saline. Use suspension of these organisms within 2-4 hours. The suspension may be stored at 4° to 8° for a validated period of time. Remove immediately a suitable sample from each suspension and determine the number of cfu per ml. in each suspension by pour plate method or filtration method. This value serves to determine the inoculum concentration and the baseline to use in the test.Procedure:
If sufficient volume (at least 20 ml) of product is available in each container and the product container can be inoculated aseptically then the test can be conducted in five original containers of the product. If filled volume is less, or the container cannot be inoculated aseptically then transfer (atleast 20 ml) the product in each of five suitable sterile containers. Inoculate each container with one of the prepared and standardized inoculum in such a way that after inoculation the final concentration of the organisms remains between 1x 10^5 and 1x 10^6 cfu per ml and the volume of the inoculum does not exceed 1 percent of the volume of the product.The initial concentration of the viable organisms in each test preparation is estimated based on the concentration of the microorganisms in each of the standardized inoculum as determined by the pour plate method or membrane filtration method. Incubate the inoculated containers at room temperature.Determine the viable count by plate-count method at 7, 14, and 28 days subsequent to the inoculation. Record any changes observed in the appearance at these intervals. From the calculated concentration of cfu per ml present at the start of the test, calculate the percentage of reduction in cfu per ml for each organism at the stated test intervals and express the changes in terms of percentage of initial concentration.
Interpretation
The preservatives are considered to be effective if:i) For parenteral, ophthalmic, sterile nasal and optic preparations:
(a) the concentration of the viable bacteria are not more than 10 percent of the initial concentration at 7 days and not more than 0.1 percent of the initial concentration at 14 days and there is a further decrease in count at 28 days.
(b) there is no increase in yeast and mold count at 7, 14 and 28 days from the initial count.
ii) For topical preparations made with aqueous base, nonsterile nasal preparation and emulsions including those applied to mucous membrane: (a) the concentration of the viable bacteria are not more than 1 percent of the initial concentration at 14 days and there is a further decrease in count at 28 days. (b) there is no increase in yeast and mold count at 14 and 28 days from the initial count.
iii) For oral preparations: (a) the concentration of the viable bacteria are not more than 10 percent of the initial concentration at 14 days and there is a further decrease in count at 28 days. (b) there is no increase in yeast and mold count at 14 and 28 days from the initial count.
dear sir,
ReplyDeletemy name is Rio, we will develop the effectiveness antimicrobial testing for our products. Can you share some examples of the protocol templates? thank you