51 ANTIMICROBIAL EFFECTIVENESS TESTING
Antimicrobial prervatives are substances added to nonsterile dosage forms to protect them from microbiological growth or from microorganisms that are introduced inadvertently during or subquent to the manufacturing process. In the ca of sterile articles packaged in multiple-do containers, antimicrobial prervatives are added to inhibit the growth of microorganisms that may be introduced from repeatedly withdrawing individual dos.
Antimicrobial prervatives should not be ud as a substitute for good manufacturing practices or solely to reduce the viable microbial population of a nonsterile product or control the presterilization bioburden of multido formulations during manufacturing. Antimicrobial prervatives in compendial dosage forms meet the requirements for Added Substances under Ingredients and Process in the General Notices.
All uful antimicrobial agents are toxic substances. For maximum protection of patients, the concentration of the prervative shown to be effective in the final packaged product should be below a level that may be toxic to human beings.
The concentration of an added antimicrobial prervative can be kept at a minimum if the active ingredients of the formulation posss an intrinsic antimicrobial activity. Antimicrobial effectiveness, whether inherent in the product or whether produced becau of the addition of an antimicrobial prervative, must be demonstrated for all injections packaged in multiple-do containers or for other products containing antimicrobial prervatives. Antimicrobial effectiveness must be demonstrated for multiple-do topical and oral dosage forms and for other dosage forms such as ophthalmic, otic, nasal, irrigation, and dialysis fluids (e Pharmaceutical Dosage Forms 1151).
This chapter provides tests to demonstrate the effectiveness of antimicrobial protection. Added antimicrobial prervatives must be declared on the label. The tests and criteria for effectiveness apply to a product in the original, unopened container in which it was distributed by the manufacturer.
PRODUCT CATEGORIES
For the purpo of testing, compendial articles have been divided into four categories (e Table 1). The criteria of antimicrobial effectiveness for the products are a function of the route of administration.
Table 1. Compendial Product Categories
Category Product Description
1Injections, other parenterals including
emulsions, otic products, sterile nasal
products, and ophthalmic products made
with aqueous bas or vehicles.
2Topically ud products made with aqueous
bas or vehicles, nonsterile nasal products,
and emulsions, including tho applied
to mucous membranes.
3Oral products other than antacids, made with
TEST ORGANISMS
U cultures of the following microorganisms 1: Candida albicans (ATCC No. 10231), Aspergillus niger (ATCC No. 16404), Escherichia coli (ATCC No. 8739), Pudomonas aeruginosa (ATCC No. 9027), and Staphylococcus aureus (ATCC No. 6538). The viable microorganisms ud in the test must not be more than five passages removed from the original ATCC culture. For purpos of the test, one passage is defined as the transfer of organisms from an established culture to fresh medium. All transfers are counted. In the ca of organisms maintained by ed-lot techniques, each cycle of freezing, thawing, and revival in fresh medium is taken as one transfer. A ed-stock technique should be ud for long-term storage of cultures. Cultures received from the ATCC should be resuscitated according to directions. If grown in broth, the cells are pelleted by centrifugation. Resuspend in 1/20th the volume of fresh maintenance
broth, and add an equal volume of 20% (v/v in water) sterile glycerol. Cells grown on agar may be scraped from the surface into the 10% glycerol broth. Dispen small aliquots of the
suspension into sterile vials. Store the vials in liquid nitrogen or in a mechanical freezer at no more than 50. When a fresh ed-stock vial is required, it may be removed and ud to inoculate a ries
of working cultures. The working cultures may then be ud periodically (each day in the ca of bacteria and yeast) to start the inoculum culture.
curtainMEDIA
All media ud in the test must be tested for growth promotion. U the microorganisms indicated above under Test Organisms.
PREPARATION OF INOCULUM
Preparatory to the test, inoculate the surface of a suitable volume of solid agar medium from a recently revived stock culture of each of the specified microorganisms. The culture conditions for the inoculum culture are described in Table 2 in which the suitable media are Soybean –Cain Digest or Sabouraud Dextro Agar Medium (e Microbial Enumeration Tests 61 and Tests for Specified Microorganisms 62).
To harvest the bacterial and C. albicans cultures, u sterile saline TS, washing the surface growth, collecting it in a suitable vesl, and adding sufficient sterile saline TS to obtain a microbial count of about 1 × 108
redis
tinyoscolony-forming units (cfu) per mL. To harvest the cells of A. niger , u sterile saline TS containing 0.05% of polysorbate 80, and add sufficient sterile saline
TS to obtain a count of about 1 × 108 cfu per mL.
Alternatively, the stock culture organisms may be grown in a suitable liquid medium (i.e., Soybean –Cain Digest Broth or Sabouraud Dextro Broth) and the cells harvested by
centrifugation, then washed and resuspended in sterile saline TS to obtain a microbial count of about 1 × 108 cfu per mL. [Note —The estimate of inoculum concentration may be performed aqueous bas or vehicles.
4
Antacids made with an aqueous ba.
by turbidimetric measurements for the challenge microorganisms. Refrigerate the suspension if it is not ud within 2 hours. ]
Determine the number of cfu per mL in each suspension, using the conditions of media and microbial recovery incubation times listed in Table 2 to confirm the initial cfu per mL estimate. This value rves to calibrate the size of inoculum ud in the test. The bacterial and yeast suspensions are to be ud within 24 hours of harvest, but the fungal preparation may be stored under refrigeration for up to 7 days.
PROCEDURE
The test can be conducted either in five original containers if sufficient volume of product is available in each container and the product container can be entered aptically (i.e., needle and syringe through an elastomeric rubber stopper), or in five sterile, capped bacteriological containers of suitable size into which a sufficient volume of product has been transferred. Inoculate each container with one of the prepared and standardized inoculum, and mix. The volume of the suspension inoculum ud is between 0.5% and 1.0% of the volume of the product. The concentration of test microorganisms that is added to the product (Categories 1, 2, and 3) are such that the final concentration of the test preparation after inoculation is
between 1 × 105 and 1 × 106 cfu per mL of the product. For Category 4 products (antacids)
the final concentration of the test preparation after inoculation is between 1 × 103 and 1 × 10拖延症基因找到了
4cfu per mL of the product.
The initial concentration of viable microorganisms in each test preparation is estimated bad on the concentration of microorganisms in each of the standardized inoculum as determined by the plate-count method.
Incubate the inoculated containers at 22.5 ± 2.5. Sample each container at the appropriate intervals specified in Table 3. Record any changes obrved in appearance at the intervals. Determine by the plate-count procedure the number of cfu prent in each test preparation for the applicable intervals (e Procedure under Microbial Enumeration Tests 61 and Tests for Specified Microorganisms 62). Incorporate an inactivator (neutralizer) of the specific antimicrobial in the plate count or in the appropriate dilution prepared for plating. The
conditions are determined in the validation study for that sample bad upon the conditions of media
and microbial recovery incubation times listed in Table 2. Using the calculated
concentrations of cfu per mL prent at the start of the test, calculate the change in log 10 values of the concentration of cfu per mL for each microorganism at the applicable test intervals, and express the changes in terms of log reductions.
经营范围英文
Table 2. Culture Conditions for Inoculum Preparation
Organism Suitable Medium Incubation Temperature Inoculum
Incubation
Time
Microbial Recovery Incubation Time Escherichia coli
(ATCC No. 8739)Soybean –Cain Digest Broth;
Soybean –Cain
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32.5 ± 2.518 to 24 hours 3 to 5 days
CRITERIA FOR ANTIMICROBIAL EFFECTIVENESS
The requirements for antimicrobial effectiveness are met if the criteria specified under Table 3 are met (e Significant Figures and Tolerances under General Notices ). No increa is defined as not more than 0.5 log 10
unit higher than the previous value measured. Table 3. Criteria for Tested Microorganisms
1 Available from American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209 (www.atcc).
男士风衣搭配Digest Agar Pudomonas
aeruginosa
(ATCC No. 9027)
Soybean –Cain Digest Broth; Soybean –Cain Digest Agar 32.5 ± 2.518 to 24 hours 3 to 5 days Staphylococcus
aureus
(ATCC No. 6538)
Soybean –Cain Digest Broth; Soybean –Cain Digest Agar 32.5 ± 2.518 to 24 hours 3 to 5 days Candida albicans
(ATCC No. 10231)
Sabouraud Dextro Agar; Sabouraud Dextro Broth 22.5 ± 2.544 to 52 hours 3 to 5 days Aspergillus niger
(ATCC No. 16404)Sabouraud Dextro Agar;
Sabouraud Dextro
Broth 22.5 ± 2.5 6 to 10 days 3 to 7 days
For Category 1 Products Bacteria:Not less than 1.0 log reduction from the initial calculated count at 7 days, not lessjob1
than 3.0 log reduction from the initial count at 14 days, and no increa from the
14 days' count at 28 days.
Yeast and Molds:No increa from the initial calculated count at 7, 14, and 28 days.
For Category 2 Products
Bacteria:Not less than 2.0 log reduction from the initial count at 14 days, and no increa
from the 14 days' count at 28 days.
Yeast and Molds:No increa from the initial calculated count at 14 and 28 days.
For Category 3 Products
Bacteria:Not less than 1.0 log reduction from the initial count at 14 days, and no increa
from the 14 days' count at 28 days.
Yeast and Molds:No increa from the initial calculated count at 14 and 28 days.
For Category 4 Products
cross overBacteria,
Yeast,
and Molds:
No increa from the initial calculated count at 14 and 28 days.
Auxiliary Information — Plea check for your question in the FAQs before contacting USP. USP34–NF29 Page 48 Topic/Question Contact
Expert Committee General Chapter Radhakrishna S Tirumalai,
猴子用英文怎么说
Ph.D.
Principal Scientific Liaison
1-301-816-8339
(GCM2010) General Chapters - Microbiology
