Usp Controles Microbiologicos

USP CONTROLES MICROBIOLOGICOS
51 ANTIMICROBIAL EFFECTIVENESS TESTING Antimicrobial preservatives are substances added to nonsterile dosage forms to protect them from microbiological growth or from microorganisms that are introduced inadvertently during or subsequent to the manufacturing process. In the case of sterile articles packaged in multiple-dose containers, antimicrobial preservatives are added to inhibit the growth of microorganisms that may be introduced from repeatedly withdrawing individual doses. Antimicrobial preservatives should not be used 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 multidose formulations during manufacturing. Antimicrobial preservatives in compendial dosage forms meet the requirements for Added Substances under Ingredients and Processes in the General Notices. All useful antimicrobial agents are toxic substances. For maximum protection of patients, the concentration of the preservative 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 preservative can be kept at a minimum if the active ingredients of the formulation possess an intrinsic antimicrobial activity. Antimicrobial effectiveness, whether inherent in the product or whether produced because of the addition of an antimicrobial preservative, must be demonstrated for all injections packaged in multiple-dose containers or for other products containing antimicrobial preservatives. Antimicrobial effectiveness must be demonstrated for multiple-dose topical and oral dosage forms and for other dosage forms such as ophthalmic, otic, nasal, irrigation, and dialysis fluids (see Pharmaceutical Dosage Forms 1151). This chapter provides tests to demonstrate the effectiveness of antimicrobial protection. Added antimicrobial preservatives 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.
1
PRODUCT CATEGORIES For the purpose of testing, compendial articles have been divided into four categories (see Table 1). The criteria of antimicrobial effectiveness for these products are a function of the route of administration. Table 1. Compendial Product Categories Category Product Description 0 Injections, other parenterals including emulsions, otic products, sterile nasal products, and ophthalmic products made with aqueous bases or vehicles. Topically used products made with aqueous bases or vehicles, nonsterile nasal products, and emulsions, including those applied to mucous membranes. Oral products other than antacids, made with aqueous bases or vehicles. Antacids made with an aqueous base. TEST ORGANISMS Use cultures of the following microorganisms1 : Candida albicans (ATCC No. 0), Aspergillus niger (ATCC No. 0), Escherichia coli (ATCC No. 0), Pseudomonas aeruginosa (ATCC No. 0), and Staphylococcus aureus (ATCC No. 0). The viable microorganisms used in the test must not be more than five passages removed from the original ATCC culture. For purposes 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 case of organisms maintained by seed lot techniques, each cycle of freezing, thawing, and revival in fresh medium is taken as one transfer. A seed stock technique should be used 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 0/0th the volume of fresh maintenance broth, and add an equal volume of 0% (v/v in water) sterile glycerol. Cells grown on agar may be scraped from the surface into the 0% glycerol broth. Dispense small aliquots of the suspension into sterile vials. Store the vials in
2
0 0

liquid nitrogen or in a mechanical freezer at no more than 0 . When a fresh seed stock vial is required, it may be removed and used to inoculate a series of working cultures. These working cultures may then be used periodically (each day in the case of bacteria and yeast) to start the inoculum culture.
MEDIA All media used in the test must be tested for growth promotion. Use 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 0 in which the suitable media are Soybean-Casein Digest or Sabouraud Dextrose Agar Medium (see Microbial Limits Testing 61). To harvest the bacterial and C. albicans cultures, use sterile saline TS, washing the surface growth, collecting it in a suitable vessel, and adding sufficient sterile saline TS to obtain a microbial count of about 0 00 colony-forming units (cfu) per mL. To harvest the cells of A. niger, use sterile saline TS containing 0% of polysorbate 0, and add sufficient sterile saline TS to obtain a count of about 0 0 0 cfu per mL. Alternatively, the stock culture organisms may be grown in a suitable liquid medium (i.e., Soybean-Casein Digest Broth or Sabouraud Dextrose Broth) and the cells harvested by centrifugation, then washed and resuspended in sterile saline TS to obtain a microbial count of about 0 00 cfu per mL. [NOTEThe estimate of inoculum concentration may be performed by turbidimetric measurements for the challenge microorganisms. Refrigerate the suspension if it is not used within 0 hours. ]

Determine the number of cfu per mL in each suspension, using the conditions of media and microbial recovery incubation times listed in Table 0 to confirm the initial cfu per mL estimate. This value serves to calibrate the size of inoculum used in the test. The bacterial and yeast suspensions are to be used within 0 hours of harvest, but the fungal preparation may be stored under refrigeration for up to seven 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 aseptically (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 used is between 0% and 0% of the volume of the product. The concentration of test microorganisms that is added to the product (Categories 0, 0, and 0) are such that the final concentration of the test preparation after inoculation is between 0 00 and 0 00 cfu per mL of the product. For Category 0 products (antacids) the final concentration of the test preparation after inoculation is between 0 00 and 0 00 cfu per mL of the product. The initial concentration of viable microorganisms in each test preparation is estimated based on the concentration of microorganisms in each of the standardized inoculum as determined by the plate-count method. Incubate the inoculated containers at 0 0 . Sample each container at the appropriate intervals specified in Table 0. Record any changes observed in appearance at these intervals. Determine by the plate-count procedure the number of cfu present in each test preparation for the applicable intervals (see Procedure under Microbial Limit Tests 61). Incorporate an inactivator (neutralizer) of the specific antimicrobial in the plate count or in the appropriate dilution prepared for plating. These conditions are determined in the validation study for that sample based upon the conditions of media and microbial recovery incubation times listed
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in Table 2. Using the calculated concentrations of cfu per mL present at the start of the test, calculate the change in log0 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. Table 2. Culture Conditions for Inoculum Preparation Microbial Recovery Incubation Time 0 to 0 days
Organism Escherichia coli (ATCC No. 0)
Suitable Medium Soybean Casein Digest Broth; Soybean Casein Digest Agar Soybean Casein Digest Broth; Soybean Casein Digest Agar Soybean Casein Digest Broth; Soybean Casein Digest Agar Sabouraud Dextrose Agar; Sabouraud Dextrose Broth Sabouraud Dextrose Agar;
Incubation Temperature 00
Inoculum Incubation Time 0 to 0 hours
Pseudomonas aeruginosa (ATCC No. 0)
00
0 to 0 hours
0 to 0 days
Staphylococcus aureus (ATCC No. 0)
00
0 to 0 hours
0 to 0 days
Candida albicans (ATCC No. 0)
00
0 to 0 hours
0 to 0 days
Aspergillus niger (ATCC No. 0)
00
0 to 0 days 0 to 0 days

Inoculum Incubation Time Microbial Recovery Incubation Time
Organism
Suitable Medium Sabouraud Dextrose Broth
Incubation Temperature
CRITERIA FOR ANTIMICROBIAL EFFECTIVENESS The requirements for antimicrobial effectiveness are met if the criteria specified under Table 3 are met (see Significant Figures and Tolerances under General Notices). No increase is defined as not more than 0 log0 unit higher than the previous value measured. Table 3. Criteria for Tested Microorganisms For Category 0 Products Bacteria: Not less than 0 log reduction from the initial calculated count at 0 days, not less than 0 log reduction from the initial count at 0 days, and no increase from the 0 days' count at 0 days. No increase from the initial calculated count at 0, 0, and 0 days. For Category 0 Products Bacteria: Yeast and Molds: Bacteria: Yeast and Molds: Not less than 0 log reduction from the initial count at 0 days, and no increase from the 0 days' count at 0 days. No increase from the initial calculated count at 0 and 0 days. For Category 0 Products Not less than 0 log reduction from the initial count at 0 days, and no increase from the 0 days' count at 0 days. No increase from the initial calculated count at 0 and 0 days. For Category 0 Products Bacteria, No increase from the initial calculated count at 0 and 0 Yeast, days. and Molds:
6
Yeast and Molds:

0
Available from American Type Culture Collection, 0 University Boulevard, Manassas, VA 0-0
(http://www.atcc.org).
55 BIOLOGICAL INDICATORSRESISTANCE PERFORMANCE TESTS
TOTAL VIABLE SPORE COUNT Remove three specimens of the relevant biological indicator from their original individual containers. Disperse the paper into component fibers by placing the test specimens in a sterile 0-mL cup of a suitable blender containing 0 mL of chilled, sterilized Purified Water and blending for 0 to 0 minutes to achieve a homogeneous suspension. Transfer a 0-mL aliquot of the suspension to a sterile, screw-capped 0- 0-mm tube. For Biological Indicator for Steam Sterilization, Paper Carrier, heat the tube containing the suspension in a water bath at 0 to 0 for 0 minutes (heat shock), starting the timing when the temperature reaches 0 . For Biological Indicator for Dry-Heat Sterilization, Paper Carrier, and for Biological Indicator for Ethylene Oxide Sterilization, Paper Carrier, heat the tube containing the suspension in a water bath at 0 to 0 for 0 minutes, starting the timing when the temperature reaches 0 . Cool rapidly in an ice water bath at 0 to 0 . Transfer two 0-mL aliquots to suitable tubes, and make appropriate serial dilutions in sterilized Purified Water, the dilutions being selected as calculated to yield preferably 0 to 0 colonies, but not less than 0, on each of a pair of plates when treated as described below. Where the biological indicator has a low spore concentration, it may be necessary to modify the dilution series and to use more plates at each dilution. Prepare a separate series of plates for each aliquot. Place 0 mL of each selected dilution in each of two 0- 0mm Petri dishes. Within 0 minutes, add to each plate 0 mL of Soybean-Casein Digest Agar Medium (see Microbial Limit Tests 61) that has been melted and cooled to 0 to 0 . Swirl to attain a homogeneous suspension, and allow to solidify. Incubate the plates in an inverted position at 0 to 0 for Biological Indicator for Steam Sterilization, Paper Carrier, and at 0 to 0 for Biological Indicator for Ethylene Oxide Sterilization, Paper Carrier, and for Biological Indicator for Dry-Heat Sterilization, Paper Carrier, or at the optimal recovery temperature specified by the manufacturer, and examine the plates after 0 and 0 hours, 7

recording for each plate the number of colonies, and using the number of colonies after 0 hours to calculate the results. Calculate the average number of spores per specimen from the results, using the appropriate dilution factor. The test is valid if the log number of spores per Carrier at 0 hours is equal to or greater than the log number after 0 hours in each case. For Biological Indicator for Steam Sterilization, Self-Contained, aseptically remove the spore strip from the container, and proceed as directed for Biological Indicator for Steam Sterilization, Paper Carrier.
D VALUE DETERMINATION For all tests described in this section, handle each test specimen with aseptic precautions, using sterilized equipment where applicable. Apparatus For Biological Indicator for Dry-Heat Sterilization, Paper Carrier, use an apparatus of known thermodynamic characteristics that has been validated for compliance with the requirements for safety1 and performance,2 that consists of a sterilizing chamber equipped with a means of heating the contained air, preferably electrically rather than gas fired, and that has adequate movement of the air through forced ventilation (by mechanical devices such as blowers), with sensing and control devices for temperature and timing capable of indicating with an accuracy of not more than 0 and 0-second intervals, respectively. The geometrical pattern of the heat source(s) is such as to enable the biological indicators under test to be uniformly heated under the specified conditions. The temperature profile in the chamber is known, and cold spots, hot spots, and slow heat zones identified. The chamber has the capability to work within a temperature range of 0 to 0 , with an accuracy at any particular setting of not less than 0 . The apparatus is equipped with a suitable additional access door or port so as to enable the entry and insertion (or removal) of specimens within 0 seconds and to enable the temperature to return to the set temperature within 0 minute where the specified temperature is 0 to 0 and within 0 minute where such temperature is 0 and above. For Biological Indicator for Ethylene Oxide Sterilization, Paper Carrier, use an apparatus that consists of a test chamber with a means of ensuring adequate mixing of the sterilant 8

gas and a means of heating the sterilant gas to not lower than the preselected operating temperature so that no liquid enters the test chamber, equipped with temperature control and monitoring, pressure control, humidification, and gas concentration monitoring devices. Detailed specifications and operational parameters for suitable apparatus are those published in Standard for a Biological IndicatorEvaluator Resistometer for Ethylene Oxide Gas Vessels (BIER/EO) Gas Vessels.3 For Biological Indicator for Steam Sterilization, Paper Carrier, and for Biological Indicator for Steam Sterilization, Self-Contained, use an apparatus that consists of a chamber equipped with heating, temperature, and steam control and monitoring devices. Detailed specifications and operational parameters for suitable apparatus are those published in Standard for a Biological IndicatorEvaluator Resistometer for Saturated Steam (BIER/Steam Vessels).4 Procedure Carry out the tests for D value at each of the applicable sets of sterilization conditions for which the packaged biological indicator under test is labeled for use. Take a sufficient number of groups of specimens of biological indicators in their original individual containers, each group consisting of 0 to 0 specimens. The number of groups provides a range of observations from not less than one labeled D value below the labeled survival time through not less than one labeled D value above the labeled kill time. Place each group on a separate suitable specimen holder that permits each specimen to be exposed to the prescribed sterilizing condition at a specific location in the sterilizing chamber. Check the apparatus for operating parameters using specimen holders without specimens. Select a series of sterilizing times in increments from the shortest time for the specimens to be tested. The differences in sterilizing times over the series are as constant as feasible, and the difference between adjacent times is no greater than 0% of the labeled D value. For Biological Indicator for Dry-Heat Sterilization, Paper Carrier, preheat the sterilizing chamber for 0 minutes. Open the access door or port, place one of the holders with a group of specimens in the sterilizing chamber, close the access door or port, and continue to operate the apparatus. Commence timing the heat exposure when the chamber temperature returns to 0 below the specified temperature. After the contents have been subjected to the sterilizing condition for a predetermined time selected from a series of time increments, remove the holder with the heated specimens, and replace it with another 9

holder with specimens. Repeat the sterilizing procedure similarly, but for another predetermined time, and continue with successive groups until all have been heated appropriately. For Biological Indicator for Ethylene Oxide Sterilization, Paper Carrier, proceed as follows: 1. Evacuate the test chamber to a pressure of not more than 0 0 mm of mercury. 2. Inject sufficient water vapor (e.g., saturated steam) to bring the chamber contents to within 0% relative humidity of the required humidification condition, and allow the chamber to equilibrate with moisture and to temperature for about 0 minutes. 3. Inject a sufficient quantity of temperature-equilibrated ethylene oxide gas to attain the appropriate concentration 0 mg of ethylene oxide per liter. 4. Subject a group of specimens to the appropriate temperature, humidification, and gas concentration conditions for the required time. 5. Evacuate the test chamber to a pressure of 0 0 mm of mercury, and release the vacuum with sterile filtered air. Repeat this until not less than 0% of the remaining gas has been removed, and remove the holder(s) with the exposed specimens. For exposing further groups of specimens to the sterilization conditions, proceed with steps 0 and 0. 6. Flush the test chamber five times with filtered air after evacuation each time to a pressure of not more than 0 0 mm of mercury. 7. Repeat the entire sterilizing procedure, steps 0 through 0, for other groups of unexposed specimens, but maintain the specified conditions of step 0 for each of the other required times. For Biological Indicator for Steam Sterilization, Paper Carrier, exhaust the sterilizing chamber, and within 0 seconds of opening the door, place one of the holders with a group of specimens in the sterilizing chamber, and operate the apparatus to heat up the chamber contents as quickly as possible. After the contents have been subjected to the sterilizing condition for a predetermined time selected from the series of time increments, exhaust the chamber as quickly as possible. Remove the holder with the heated specimens, and replace it with another group of specimens. Repeat the sterilizing procedure similarly, but for another predetermined time, and continue with successive groups until all have been appropriately heated. 10

For Biological Indicator for Steam Sterilization, Self-Contained, follow the procedure indicated for Biological Indicator for Steam Sterilization, Paper Carrier, but handle each self-contained unit as a biological indicator system, with the D value determined for the self-contained system. Recovery After completion of the sterilizing procedure for Biological Indicator for Dry-Heat Sterilization, Paper Carrier; Biological Indicator for Ethylene Oxide Sterilization, Paper Carrier; or Biological Indicator for Steam Sterilization, Paper Carrier, whichever is applicable, and within a noted time not more than 0 hours, aseptically remove and add each strip to 0 to 0 mL of Soybean-Casein Digest Medium (see Media under Sterility Tests 71) to submerge the biological indicator completely in a suitable tube. For each Biological Indicator for Steam Sterilization, Self-Contained specimen, the paper strip is immersed in the self-contained medium according to manufacturers' instructions, within a noted time not more than 0 hours. Incubate each tube at a temperature of 0 to 0 for Biological Indicator for Steam Sterilization, Paper Carrier, and Biological Indicator for Steam Sterilization, Self-Contained, or at 0 to 0 for Biological Indicator for Dry-Heat Sterilization, Paper Carrier, and Biological Indicator for Ethylene Oxide Sterilization, Paper Carrier, or in any case at the optimal recovery temperature specified by the manufacturer. Observe each inoculated medium-containing tube at 0 and 0 hours, and every 0 or 0 days thereafter for a total of 0 days after inoculation. (Where growth is observed at any particular observation time, further incubation of the specimen(s) concerned may be omitted.) Note the number of specimens showing no evidence of growth at any time. Calculation This chapter describes the use of the Limited Spearman-Karber Method for determining the D value of biological indicators on spore paper carriers. Use this method in the event of a compendial issue or regulatory referee testing of a biological indicator system. It is recognized that other methods, such as the Survival Curve Method and the StumboMurphy-Cochran procedure, may be routinely used by manufacturers and users of biological indicators to determine D values. The calculation of the D value using the Limited Spearman-Karber Method is based on the use of 0 biological indicators per group. [NOTEIf less than 0 biological indicators are used (i.e., 0), the formula and the various 11

calculation steps will have to be modified, including the Replacement of Missing Values; however, the requirements of the test remain the same. ] Designate the number of specimens taken for each group (i.e., 0) by n, and the difference between adjacent times (in minutes) by . Designate for each group of the series the number of specimens showing no growth by: f0, f0, ... fk, in which f0 is the response of all 0 specimens showing growth (0/0 inactivated) in the group held for the shortest time for such result that is adjacent to an intermediate mortality; and fk is the response of all 0 specimens of the group showing no growth (0/0 inactivated) in the group held for the longest time for such result that is adjacent to an intermediate mortality. Do not use for the calculations observations for groups beyond the ends of the series, f0 and fk, giving results that are not adjacent to an intermediate mortality. The test is valid if there is available a result (0/0) from a group held for a shorter time than that for the selected shortest time result (f0), and there is available a result (0/0) from a group held for a longer time than that for the selected longest time result (fk). Calculate the mean heating time, T, for achieving complete kill by the equation:
in which Tk is the time for achieving the result fk. Calculate the D value by the equation:
in which N0 is the average spore count per carrier determined by Total Viable Spore Count (see above) at the time of making this test. Calculate the variance of T, VT , by the equation:
12

in which represents a constant interval between successive exposures, as defined above. The standard deviation, sT , is the square root of the variance:
Calculate the lower and upper 0% confidence limits (approximate CL) for the D value by the equation: approximate CL for D = (T 0sT/log N0 + 0). Replacement of Missing Values If not more than 0 specimen from a group and not more than 0 specimens from all of the groups giving the results f0 through fk are missing, replace each missing value by adding 0 to the number showing no growth, if the number showing no growth in the remaining 0 specimens of that group is 0 or less, and adding 0 if the number showing no growth in the remaining 0 specimens of that group is 0 or more. Survival Time and Kill Time Take two groups, each consisting of 0 specimens of the relevant biological indicator, in their original, individual containers. Place the specimens of a group in suitable specimen holders that permit each specimen to be exposed to the sterilizing conditions at a specific location in the sterilizing chamber. Check the chamber for operating parameters by preheating it to the selected temperature 0 in the cases of Biological Indicator for DryHeat Sterilization, Paper Carrier, and Biological Indicator for Ethylene Oxide Sterilization, Paper Carrier, or 0 in the cases of Biological Indicator for Steam Sterilization, Paper Carrier, and Biological Indicator for Steam Sterilization, Self-Contained. For Biological Indicator for Dry-Heat Sterilization, Paper Carrier, preheat the unit to temperature, and equilibrate the heat chamber. Open the access door or port, and place the holder(s) in the chamber, close the access door or port, and continue to operate the 13

apparatus. Commence timing the heat exposure when the chamber temperature returns to the lower limit of the selected temperature. Expose the specimens for the required survival time, enter the chamber, and remove the holder(s) containing the 0 specimens. Repeat the above procedure immediately, or preheat if a substantial interval has elapsed, so as to subject the second holder(s) containing 0 specimens similarly to the first conditions, but for the required kill time. For Biological Indicator for Ethylene Oxide Sterilization, Paper Carrier, preheat the chamber to equilibrium at the selected temperature 0 , and initiate and monitor the operating steps 0 through 0 as described for D Value Determination, appropriate for the combination of gas concentration, temperature, and relative humidity, using a gassing time in step 0 appropriate to the survival time. Repeat the above procedure with two or more groups each consisting of 0 specimens, but using a gassing time in step 0 appropriate to the kill time. For Biological Indicator for Steam Sterilization, Paper Carrier, and Biological Indicator for Steam Sterilization, Self-Contained, exhaust the steam chamber, and open the door. Within 0 seconds of opening the door place the loaded holder(s) into the chamber, and operate the apparatus to heat the chamber contents as quickly as possible. Expose the specimens for the required survival time, counting the exposure from the time when the temperature record shows that the chamber has reached the required temperature. Exhaust the chamber as quickly as possible at the end of the exposure period. When the chamber can be safely entered, remove the holder(s) containing the specimens. Repeat the above procedure immediately, or preheat if a substantial interval has elapsed, so as to subject the holder(s) containing 0 specimens similarly to the first exposure. Repeat the above procedure with two more groups each consisting of 0 specimens, but expose the specimens for the required kill time. In each case for the Biological Indicator for Dry-Heat Sterilization, Paper Carrier; Biological Indicator for Ethylene Oxide Sterilization, Paper Carrier; or Biological Indicator for Steam Sterilization, Paper Carrier, whichever is appropriate, after completion of the sterilizing procedure, and within a noted time not more than 0 hours, aseptically remove and add each carrier to 0 to 0 mL of Soybean-Casein Digest Medium (see Media under Sterility Tests 71) to submerge the biological indicator completely in a suitable tube. Incubate each tube at a temperature of 0 to 0 in the case of Biological Indicator for Steam Sterilization, Paper Carrier, or of 0 to 0 in the cases of 14

Biological Indicator for Dry-Heat Sterilization, Paper Carrier, and Biological Indicator for Ethylene Oxide Sterilization, Paper Carrier, or at the optimal temperature specified by the manufacturer. For Biological Indicator for Steam Sterilization, Self-Contained, the paper strip is immersed in self-contained medium according to manufacturers' instructions within a noted time not more than 0 hours and incubated at 0 to 0 . Observe each inoculated medium-containing tube at 0 and 0 hours, and every 0 or 0 days thereafter for a total of 0 days after inoculation. (Where growth is observed at any particular observation time, further incubation of the specimen(s) concerned may be omitted.) Note the specimens showing no evidence of growth at any time.
Safety includes design to prevent electric shock or gas exposition and burns, where operators can
wear protective clothing and gloves against burns from touching hot surfaces.
0
Descriptions of different types of dry-heat sterilizing equipment and detailed guidelines for
determining, monitoring, and controlling the operating parameters have been published by the Health Industry Manufacturers Association in Report No. 0-0, Operator Training for Dry Heat Sterilizing Equipment, and by the Parenteral Drug Association, Inc., in Technical Report No. 0, Validation of Dry Heat Processes Used for Sterilization and Depyrogenation .
0
Standard for BIER/EO Gas Vessels, 0 July 0, Association for the Advancement of Medical
Instrumentation (AAMI), 0 Washington Boulevard, Suite 0, Arlington, VA 0-0.

0
Standard for BIER/Steam Vessels, 0 July 0, AAMI, 0 Washington Boulevard, Suite 0, Arlington,
VA 0-0.
61 MICROBIAL LIMIT TESTS This chapter provides tests for the estimation of the number of viable aerobic microorganisms present and for freedom from designated microbial species in pharmaceutical articles of all kinds, from raw materials to the finished forms. An automated method may be substituted for the tests presented here, provided it has been properly validated as giving equivalent or better results. In preparing for and in applying the tests, observe aseptic precautions in handling the specimens. Unless otherwise directed, where 15

the procedure specifies simply incubate, hold the container in air that is thermostatically controlled at a temperature between 0 and 0 , for a period of 0 to 0 hours. The term growth is used in a special sense herein, i.e., to designate the presence and presumed proliferation of viable microorganisms.
Preparatory Testing The validity of the results of the tests set forth in this chapter rests largely upon the adequacy of a demonstration that the test specimens to which they are applied do not, of themselves, inhibit the multiplication, under the test conditions, of microorganisms that may be present. Therefore, preparatory to conducting the tests on a regular basis and as circumstances require subsequently, inoculate diluted specimens of the material to be tested with separate viable cultures of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Salmonella. This can be done by adding 0 mL of not less than 00 dilution of a 0-hour broth culture of the microorganism to the first dilution (in pH 0 Phosphate Buffer, Fluid Soybean-Casein Digest Medium, or Fluid Lactose Medium) of the test material and following the test procedure. Failure of the organism(s) to grow in the relevant medium invalidates that portion of the examination and necessitates a modification of the procedure by (0) an increase in the volume of diluent, the quantity of test material remaining the same, or by (0) the incorporation of a sufficient quantity of suitable inactivating agent(s) in the diluents, or by (0) an appropriate combination of modifications (0) and (0) so as to permit growth of the inocula. The following are examples of ingredients and their concentrations that may be added to the culture medium to neutralize inhibitory substances present in the sample: soy lecithin, 0%; and polysorbate 0, 0%. Alternatively, repeat the test as described in the preceding paragraph, using Fluid Casein DigestSoy LecithinPolysorbate 0 Medium to demonstrate neutralization of preservatives or other antimicrobial agents in the test material. Where inhibitory substances are contained in the product and the latter is soluble, a suitable, validated adaptation of a procedure set forth in the section Membrane Filtration Method under Test Procedures under Sterility Tests 71, may be used. If in spite of the incorporation of suitable inactivating agents and a substantial increase in the volume of diluent it is still not possible to recover the viable cultures described above and where the article is not suitable for employment of membrane filtration, it can be 16

assumed that the failure to isolate the inoculated organism is attributable to the bactericidal activity of the product. This information serves to indicate that the article is not likely to be contaminated with the given species of microorganism. Monitoring should be continued in order to establish the spectrum of inhibition and bactericidal activity of the article.
Buffer Solution and Media Culture media may be prepared as follows, or dehydrated culture media may be used provided that, when reconstituted as directed by the manufacturer or distributor, they have similar ingredients and/or yield media comparable to those obtained from the formulas given herein. In preparing media by the formulas set forth herein, dissolve the soluble solids in the water, using heat, if necessary, to effect complete solution, and add solutions of hydrochloric acid or sodium hydroxide in quantities sufficient to yield the desired pH in the medium when it is ready for use. Determine the pH at 0 0 . Where agar is called for in a formula, use agar that has a moisture content of not more than 0%. Where water is called for in a formula, use Purified Water.
PH 7.2 PHOSPHATE BUFFER
Stock Solution Dissolve 0 g of monobasic potassium phosphate in about 0 mL of water contained in a 0-mL volumetric flask. Adjust to pH 0 0 by the addition of sodium hydroxide TS (about 0 mL), add water to volume, and mix. Dispense and sterilize. Store under refrigeration. For use, dilute the Stock Solution with water in the ratio of 0 to 0, and sterilize.
MEDIA
Unless otherwise indicated, the media should be sterilized by heating in an autoclave (see Steam Sterilization under Sterilization 1211), the exposure time depending on the volume to be sterilized. I. Fluid Casein DigestSoy LecithinPolysorbate 20 Medium
17

Pancreatic Digest of Casein 0 g Soy Lecithin Polysorbate 0 Water 0g 0 mL 0 mL
Dissolve the pancreatic digest of casein and soy lecithin in 0 mL of water, heating in a water bath at 0 to 0 for about 0 minutes to effect solution. Add 0 mL of polysorbate 0. Mix, and dispense as desired. II. SoybeanCasein Digest Agar Medium Pancreatic Digest of Casein 0g
Papaic Digest of Soybean Meal 0 g Sodium Chloride Agar Water pH after sterilization: 0 0. III. Fluid SoybeanCasein Digest Medium Prepare as directed for Soybean-Casein Digest Medium under Sterility Tests 71. IV. MannitolSalt Agar Medium Pancreatic Digest of Casein 0g 0g 0g 0 mL
Peptic Digest of Animal Tissue 0 g Beef Extract

D-Mannitol
0g 0g 0g
Sodium Chloride
18

Agar Phenol Red Water 0g 0g 0 mL
Mix, then heat with frequent agitation, and boil for 0 minute to effect solution. pH after sterilization: 0 0. V. BairdParker Agar Medium Pancreatic Digest of Casein 0 g Beef Extract Yeast Extract Lithium Chloride Agar Glycine Sodium Pyruvate Water 0g 0g 0g 0g 0g 0g 0 mL
Heat with frequent agitation, and boil for 0 minute. Sterilize, cool to between 0 and 0 , and add 0 mL of sterile potassium tellurite solution (0 in 0) and 0 mL of egg-yolk emulsion. Mix intimately but gently, and pour into plates. (Prepare the egg-yolk emulsion by disinfecting the surface of whole shell eggs, aseptically cracking the eggs, and separating out intact yolks into a sterile graduated cylinder. Add sterile saline TS to obtain a 0 to 0 ratio of egg yolk to saline. Add to a sterile blender cup, and mix at high speed for 0 seconds.) pH after sterilization: 0 0. VI. VogelJohnson Agar Medium Pancreatic Digest of Casein 0g
19

Yeast Extract Mannitol 0g 0g
Dibasic Potassium Phosphate 0 g Lithium Chloride Glycine Agar Phenol Red Water 0g 0g 0g 0 mg 0 mL
Boil the solution of solids for 0 minute. Sterilize, cool to between 0 and 0 , and add 0 mL of sterile potassium tellurite solution (0 in 0). pH after sterilization: 0 0. VII. Cetrimide Agar Medium Pancreatic Digest of Gelatin Magnesium Chloride Potassium Sulfate Agar 0g 0g 0g 0g
Cetyl Trimethylammonium Bromide (Cetrimide) 0 g Glycerin Water 0 mL 0 mL
Dissolve all solid components in the water, and add the glycerin. Heat, with frequent agitation, and boil for 0 minute to effect solution. pH after sterilization: 0 0. VIII. Pseudomonas Agar Medium for Detection of Fluorescin
20

Pancreatic Digest of Casein Peptic Digest of Animal Tissue 0g 0g
Anhydrous Dibasic Potassium Phosphate 0 g Magnesium Sulfate (MgSO00H0O) Glycerin Agar Water 0g 0 mL 0g 0 mL
Dissolve the solid components in the water before adding the glycerin. Heat, with frequent agitation, and boil for 0 minute to effect solution. pH after sterilization: 0 0. IX. Pseudomonas Agar Medium for Detection of Pyocyanin Pancreatic Digest of Gelatin 0g
Anhydrous Magnesium Chloride 0 g Anhydrous Potassium Sulfate Agar Glycerin Water 0g 0g 0 mL 0 mL
Dissolve the solid components in the water before adding the glycerin. Heat, with frequent agitation, and boil for 0 minute to effect solution. pH after sterilization: 0 0. X. Fluid Lactose Medium Beef Extract 0g
Pancreatic Digest of Gelatin 0 g
21

Lactose Water Cool as quickly as possible after sterilization. pH after sterilization: 0 0. XI. Fluid SeleniteCystine Medium Pancreatic Digest of Casein 0 g Lactose Sodium Phosphate Sodium Acid Selenite
L-Cystine
0g 0 mL
0g 0g 0g 0 mg 0 mL
Water Final pH: 0 0.
Mix, and heat to effect solution. Heat in flowing steam for 0 minutes. Do not sterilize. XII. Fluid Tetrathionate Medium Pancreatic Digest of Casein 0g
Peptic Digest of Animal Tissue 0 g Bile Salts Calcium Carbonate Sodium Thiosulfate Water 0g 0g 0g 0 mL
Heat the solution of solids to boiling. On the day of use, add a solution prepared by dissolving 0 g of potassium iodide and 0 g of iodine in 0 mL of water. Then add 0 mL of a solution of brilliant green (0 in 0), and mix. Do not heat the medium after adding the brilliant green solution. 22

XIII. Brilliant Green Agar Medium Yeast Extract 0g
Peptic Digest of Animal Tissue 0 g Pancreatic Digest of Casein Lactose Sodium Chloride Sucrose Phenol Red Agar Brilliant Green Water 0g 0g 0g 0g 0 mg 0g 0 mg 0 mL
Boil the solution of solids for 0 minute. Sterilize just prior to use, melt the medium, pour into petri dishes, and allow to cool. pH after sterilization: 0 0. XIV. XyloseLysineDesoxycholate Agar Medium Xylose
L-Lysine
0g 0g 0g 0g 0g 0g 0 mg 0g 23
Lactose Sucrose Sodium Chloride Yeast Extract Phenol Red Agar

Sodium Desoxycholate Sodium Thiosulfate 0g 0g
Ferric Ammonium Citrate 0 mg Water Final pH: 0 0. Heat the mixture of solids and water, with swirling, just to the boiling point. Do not overheat or sterilize. Transfer at once to a water bath maintained at about 0 , and pour into plates as soon as the medium has cooled. XV. Bismuth Sulfite Agar Medium Beef Extract Pancreatic Digest of Casein 0g 0g 0 mL
Peptic Digest of Animal Tissue 0 g Dextrose Sodium Phosphate Ferrous Sulfate Bismuth Sulfite Indicator Agar Brilliant Green Water Final pH: 0 0. Heat the mixture of solids and water, with swirling, just to the boiling point. Do not overheat or sterilize. Transfer at once to a water bath maintained at about 0 , and pour into plates as soon as the medium has cooled. XVI. Triple SugarIronAgar Medium 24 0g 0g 0 mg 0g 0g 0 mg 0 mL

Pancreatic Digest of Casein 0g
Pancreatic Digest of Animal Tissue 0 g Lactose Sucrose Dextrose Ferrous Ammonium Sulfate Sodium Chloride Sodium Thiosulfate Agar Phenol Red Water pH after sterilization: 0 0. XVII. MacConkey Agar Medium Pancreatic Digest of Gelatin Pancreatic Digest of Casein 0g 0g 0g 0g 0g 0 mg 0g 0 mg 0g 0 mg 0 mL
Peptic Digest of Animal Tissue 0 g Lactose Bile Salts Mixture Sodium Chloride Agar Neutral Red Crystal Violet 0g 0g 0g 0g 0 mg 0 mg
25

Water 0 mL
Boil the mixture of solids and water for 0 minute to effect solution. pH after sterilization: 0 0. XVIII. Levine EosinMethylene Blue Agar Medium Pancreatic Digest of Gelatin 0 g Dibasic Potassium Phosphate 0 g Agar Lactose Eosin Y Methylene Blue Water 0g 0g 0 mg 0 mg 0 mL
Dissolve the pancreatic digest of gelatin, the dibasic potassium phosphate, and the agar in the water, with warming, and allow to cool. Just prior to use, liquefy the gelled agar solution, add the remaining ingredients, as solutions, in the following amounts, and mix: for each 0 mL of the liquefied agar solution0 mL of lactose solution (0 in 0), 0 mL of the eosin Y solution (0 in 0), and 0 mL of methylene blue solution (0 in 0). The finished medium may not be clear. pH after sterilization: 0 0. XIX. Sabouraud Dextrose Agar Medium Dextrose Mixture of equal parts of Peptic Digest of Animal Tissue and Pancreatic Digest of Casein Agar Water 0g
0g 0g 0 mL
26

Mix, and boil to effect solution. pH after sterilization: 0 0. XX. Potato Dextrose Agar Medium Cook 0 g of peeled and diced potatoes in 0 mL of water prepared by distillation, filter through cheesecloth, add water prepared by distillation to make 0 mL, and add the following: Agar Glucose Dissolve by heating, and sterilize. pH after sterilization: 0 0. For use, just prior to pouring the plates, adjust the melted and cooled to 0 medium with sterile tartaric acid solution (0 in 0) to a pH of 0 0. Do not reheat the pH 0 medium. 0g 0g
Sampling Provide separate 0-mL or 0-g specimens for each of the tests called for in the individual monograph.
Procedure Prepare the specimen to be tested, by treatment that is appropriate to its physical characteristics and that does not alter the number and kind of microorganisms originally present, in order to obtain a solution or suspension of all or part of it in a form suitable for the test procedure(s) to be carried out. For a solid that dissolves to an appreciable extent but not completely, reduce the substance to a moderately fine powder, suspend it in the vehicle specified, and proceed as directed under Total Aerobic Microbial Count, and under Test for Staphylococcus aureus and Pseudomonas aeruginosa and Test for Salmonella species and Escherichia coli.
27

For a fluid specimen that consists of a true solution, or a suspension in water or a hydroalcoholic vehicle containing less than 0 percent of alcohol, and for a solid that dissolves readily and practically completely in 0 mL of pH 0 Phosphate Buffer or the media specified, proceed as directed under Total Aerobic Microbial Count, and under Test for Staphylococcus aureus and Pseudomonas aeruginosa and Test for Salmonella species and Escherichia coli. For water-immiscible fluids, ointments, creams, and waxes, prepare a suspension with the aid of a minimal quantity of a suitable, sterile emulsifying agent (such as one of the polysorbates), using a mechanical blender and warming to a temperature not exceeding 0 , if necessary, and proceed with the suspension as directed under Total Aerobic Microbial Count, and under Test for Staphylococcus aureus and Pseudomonas aeruginosa and Test for Salmonella species and Escherichia coli. For a fluid specimen in aerosol form, chill the container in an alcohol-dry ice mixture for approximately 0 hour, cut open the container, allow it to reach room temperature, permit the propellant to escape, or warm to drive off the propellant if feasible, and transfer the quantity of test material required for the procedures specified in one of the two preceding paragraphs, as appropriate. Where 0 g or 0 mL of the specimen, whichever is applicable, cannot be obtained from 0 containers in aerosol form, transfer the entire contents from 0 chilled containers to the culture medium, permit the propellant to escape, and proceed with the test on the residues. If the results of the test are inconclusive or doubtful, repeat the test with a specimen from 0 more containers. Total Aerobic Microbial Count For specimens that are sufficiently soluble or translucent to permit use of the Plate Method, use that method; otherwise, use the Multiple-Tube Method. With either method, first dissolve or suspend 0 g of the specimen if it is a solid, or 0 mL, accurately measured, if the specimen is a liquid, in pH 0 Phosphate Buffer, Fluid SoybeanCasein Digest Medium, or Fluid Casein DigestSoy Lecithin-Polysorbate 0 Medium to make 0 mL. For viscous specimens that cannot be pipeted at this initial 0:0 dilution, dilute the specimen until a suspension is obtained, i.e., 0:0 or 0:0, etc., that can be pipeted. Perform the test for absence of inhibitory (antimicrobial) properties as described under Preparatory Testing before the determination of Total Aerobic Microbial Count. Add the specimen to the medium not more than 0 hour after preparing the appropriate dilutions for inoculation. 28

PLATE METHOD Dilute further, if necessary, the fluid so that 0 mL will be expected to
yield between 0 and 0 colonies. Pipet 0 mL of the final dilution onto each of two sterile petri dishes. Promptly add to each dish 0 to 0 mL of SoybeanCasein Digest Agar Medium that previously has been melted and cooled to approximately 0 . Cover the petri dishes, mix the sample with the agar by tilting or rotating the dishes, and allow the contents to solidify at room temperature. Invert the petri dishes, and incubate for 0 to 0 hours. Following incubation, examine the plates for growth, count the number of colonies, and express the average for the two plates in terms of the number of microorganisms per g or per mL of specimen. If no microbial colonies are recovered from the dishes representing the initial 0:0 dilution of the specimen, express the results as less than 0 microorganisms per g or per mL of specimen.
MULTIPLE-TUBE METHOD Into each of fourteen test tubes of similar size place 0 mL of
sterile Fluid SoybeanCasein Digest Medium. Arrange twelve of the tubes in four sets of three tubes each. Put aside one set of three tubes to serve as the controls. Into each of three tubes of one set (0) and into a fourth tube (A) pipet 0 mL of the solution or suspension of the specimen, and mix. From tube A, pipet 0 mL of its contents into the one remaining tube (B) not included in a set, and mix. These two tubes contain 0 mg (or 0 L) and 0 mg (or 0 L) of the specimen, respectively. Into each of the second set (0) of three tubes pipet 0 mL from tube A, and into each tube of the third set (0) pipet 0 mL from tube B. Discard the unused contents of tubes A and B. Close well, and incubate all of the tubes. Following the incubation period, examine the tubes for growth: the three control tubes remain clear and the observations in the tubes containing the specimen, when interpreted by reference to Table 1, indicate the most probable number of microorganisms per g or per mL of specimen. Table 1. Most Probable Total Count by Multiple-Tube Method Observed Combinations of Numbers of Tubes Showing Growth in Each Set No. of mg (or mL) of Specimen per Tube 0 (0 L) 0 (0 L) 0 (0 L) Most Probable Number of Microorganisms per g or per mL
29

Observed Combinations of Numbers of Tubes Showing Growth in Each Set No. of mg (or mL) of Specimen per Tube 0 (0 L) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 (0 L) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 (0 L) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Most Probable Number of Microorganisms per g or per mL >0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Test for Staphylococcus aureus and Pseudomonas aeruginosa To the specimen add Fluid SoybeanCasein Digest Medium to make 0 mL, mix, and incubate. Examine the 30

medium for growth, and if growth is present, use an inoculating loop to streak a portion of the medium on the surface of VogelJohnson Agar Medium (or BairdParker Agar Medium, or MannitolSalt Agar Medium) and of Cetrimide Agar Medium, each plated on petri dishes. Cover and invert the dishes, and incubate. If, upon examination, none of the plates contains colonies having the characteristics listed in Tables 2 and 3 for the media used, the test specimen meets the requirements for freedom from Staphylococcus aureus and Pseudomonas aeruginosa. Table 2. Morphologic Characteristics of Staphylococcus aureus on Selective Agar Media Selective Medium Vogel-Johnson Agar Medium
Characteristic Colonial Morphology Black Surrounded by yellow zone
Gram Stain Positive cocci (in clusters) Positive cocci (in clusters) Positive cocci (in clusters)
Mannital-Salt Agar Medium
Yellow colonies with yellow zones
Baird-Parker Agar Medium
Black, shiny, surrounded by clear zones 0 to 0 mm
Table 3. Morphologic Characteristics of Pseudomonas aeruginosa on Selective and Diagnostic Agar Media Characteristic Colonial Morphology Generally greenish Fluorescence in Oxidase UV Light Test Greenish Positive
Selective Medium Centrimide Agar Medium Pseudomonas Agar Medium for Detection of Fluorescin
Gram Stain Negative rods Negative rods
Generally colorless to yellowish
Yellowish
Positive
31

Characteristic Colonial Morphology Generally greenish Fluorescence in Oxidase UV Light Test Blue Positive Gram Stain Negative rods
Selective Medium Pseudomonas Agar Medium for Detection of Pyocyanin
COAGULASE TEST (FOR Staphylococcus aureus) With the aid of an inoculating loop,
transfer representative suspect colonies from the agar surfaces of the VogelJohnson Agar Medium (or BairdParker Agar Medium, or MannitolSalt Agar Medium) to individual tubes, each containing 0 mL of mammalian, preferably rabbit or horse, plasma with or without suitable additives. Incubate in a water bath at 0 , examining the tubes at 0 hours and subsequently at suitable intervals up to 0 hours. Test positive and negative controls simultaneously with the unknown specimens. If no coagulation in any degree is observed, the specimen meets the requirements of the test for absence of Staphylococcus aureus.
OXIDASE AND PIGMENT TESTS (FOR Pseudomonas aeruginosa) With the aid of an
inoculating loop, streak representative suspect colonies from the agar surface of Cetrimide Agar Medium on the agar surfaces of Pseudomonas Agar Medium for Detection of Fluorescin and Pseudomonas Agar Medium for Detection of Pyocyanin contained in petri dishes. If numerous colonies are to be transferred, divide the surface of each plate into quadrants, each of which may be inoculated from a separate colony. Cover and invert the inoculated media, and incubate at 0 0 for not less than three days. Examine the streaked surfaces under UV light. Examine the plates to determine whether colonies having the characteristics listed in Table 3 are present. Confirm any suspect colonial growth on one or more of the media as Pseudomonas aeruginosa by means of the oxidase test. Upon the colonial growth place or transfer colonies to strips or disks of filter paper that previously has been impregnated with N,Ndimethyl-p-phenylenediamine dihydrochloride: if there is no development of a pink color, changing to purple, the specimen meets the requirements of the test for the absence of Pseudomonas aeruginosa. The presence of Pseudomonas aeruginosa may be confirmed by other suitable cultural and biochemical tests, if necessary.
32

Test for Salmonella species and Escherichia coli To the specimen, contained in a suitable vessel, add a volume of Fluid Lactose Medium to make 0 mL, and incubate. Examine the medium for growth, and if growth is present, mix by gently shaking. Pipet 0mL portions into vessels containing, respectively, 0 mL of Fluid SeleniteCystine Medium and Fluid Tetrathionate Medium, mix, and incubate for 0 to 0 hours. (Retain the remainder of the Fluid Lactose Medium.)
TEST FOR Salmonella SPECIES By means of an inoculating loop, streak portions from
both the selenite-cystine and tetrathionate media on the surface of Brilliant Green Agar Medium, XyloseLysineDesoxycholate Agar Medium, and Bismuth Sulfite Agar Medium contained in petri dishes. Cover and invert the dishes, and incubate. Upon examination, if none of the colonies conforms to the description given in Table 4, the specimen meets the requirements of the test for absence of the genus Salmonella. Table 4. Morphologic Characteristics of Salmonella Species on Selective Agar Media Selective Medium Brilliant Green Agar Medium Xylose-LysineDesoxycholate Agar Medium Characteristic Colonial Morphology
Small, transparent, colorless or pink to white opaque (frequently surrounded by pink to red zone) Red, with or without black centers
Bismuth Sulfite Black or green Agar Medium If colonies of Gram-negative rods matching the description in Table 4 are found, proceed with further identification by transferring representative suspect colonies individually, by means of an inoculating wire, to a butt-slant tube of Triple SugarIronAgar Medium by first streaking the surface of the slant and then stabbing the wire well beneath the surface. Incubate. If examination discloses no evidence of tubes having alkaline (red) slants and acid (yellow) butts (with or without concomitant blackening of the butt from hydrogen sulfide production), the specimen meets the requirements of the test for the absence of the genus Salmonella.*
33

TEST FOR Escherichia coli By means of an inoculating loop, streak a portion from the
remaining Fluid Lactose Medium on the surface of MacConkey Agar Medium. Cover and invert the dishes, and incubate. Upon examination, if none of the colonies conforms to the description given in Table 5 for this medium, the specimen meets the requirements of the test for absence of Escherichia coli. Table 5. Morphologic Characteristics of Escherichia coli on MacConkey Agar Medium Gram Stain Characteristic Colonial Morphology
Negative rods Brick-red; may have surrounding zone of precipitated bile (cocco-bacilli) If colonies matching the description in Table 5 are found, proceed with further identification by transferring the suspect colonies individually, by means of an inoculating loop, to the surface of Levine EosinMethylene Blue Agar Medium, plated on petri dishes. If numerous colonies are to be transferred, divide the surface of each plate into quadrants, each of which may be seeded from a separate colony. Cover and invert the plates, and incubate. Upon examination, if none of the colonies exhibits both a characteristic metallic sheen under reflected light and a blue-black appearance under transmitted light, the specimen meets the requirements of the test for the absence of Escherichia coli. The presence of Escherichia coli may be confirmed by further suitable cultural and biochemical tests. Total Combined Molds and Yeasts Count Proceed as for the Plate Method under Total Aerobic Microbial Count, except for using the same amount of Sabouraud Dextrose Agar Medium or Potato Dextrose Agar Medium, instead of Soybean Casein Digest Medium, and except for incubating the inverted petri dishes for 0 to 0 days at 0 to 0 . Retest For the purpose of confirming a doubtful result by any of the procedures outlined in the foregoing tests following their application to a 0-g specimen, a retest on a 0-g specimen of the product may be conducted. Proceed as directed under Procedure, but make allowance for the larger specimen size.
Additional, confirmatory evidence may be obtained by use of procedures set forth in Official
Methods of Analysis of the AOAC, 0th ed. (0), sections 0-0.
34

Change to read: 71 STERILITY TESTS
Portions of this general chapter have been harmonized with the corresponding texts of the European Pharmacopeia and/or the Japanese Pharmacopeia. Those portions that are not harmonized are marked with symbols ( ) to specify this fact.
The following procedures are applicable for determining whether a Pharmacopeial article purporting to be sterile complies with the requirements set forth in the individual monograph with respect to the test for sterility. Pharmacopeial articles are to be tested by the Membrane Filtration method under Test for Sterility of the Product to be Examined where the nature of the product permits. If the membrane filtration technique is unsuitable, use the Direct Inoculation of the Culture Medium method under Test for Sterility of the Product to be Examined. All devices, with the exception of Devices with Pathways Labeled Sterile, are tested using the Direct Inoculation of the Culture Medium method. Provisions for retesting are included under Observation and Interpretation of Results. Because sterility testing is a very exacting procedure, where asepsis of the procedure must be ensured for a correct interpretation of results, it is important that personnel be properly trained and qualified. The test for sterility is carried out under aseptic conditions. In order to achieve such conditions, the test environment has to be adapted to the way in which the sterility test is performed. The precautions taken to avoid contamination are such that they do not affect any microorganisms that are to be revealed in the test. The working conditions in which the tests are performed are monitored regularly by appropriate sampling of the working area and by carrying out appropriate controls. These Pharmacopeial procedures are not by themselves designed to ensure that a batch of product is sterile or has been sterilized. This is accomplished primarily by validation of the sterilization process or of the aseptic processing procedures. When evidence of microbial contamination in the article is obtained by the appropriate Pharmacopeial method, the result so obtained is conclusive evidence of failure of the article to meet the requirements of the test for sterility, even if a different result is obtained
35

by an alternative procedure. For additional information on sterility testing, see
Sterilization and Sterility Assurance of Compendial Articles 1211.
MEDIA Prepare media for the tests as described below, or dehydrated formulations may be used provided that, when reconstituted as directed by the manufacturer or distributor, they meet the requirements of the Growth Promotion Test of Aerobes, Anaerobes, and Fungi. Media are sterilized using a validated process. The following culture media have been found to be suitable for the test for sterility. Fluid Thioglycollate Medium is primarily intended for the culture of anaerobic bacteria. However, it will also detect aerobic bacteria. SoybeanCasein Digest Medium is suitable for the culture of both fungi and aerobic bacteria. Fluid Thioglycollate Medium
L-Cystine
0g 0g 0/0 g
Sodium Chloride Dextrose (C0H0O0H0O)
Agar, granulated (moisture content not exceeding 0%) 0g Yeast Extract (water-soluble) Pancreatic Digest of Casein Sodium Thioglycollate or Thioglycolic Acid Resazurin Sodium Solution (0 in 0), freshly prepared Purified Water 0g 0g 0g 0 mL
0 mL 0 mL
36

Mix the L-cystine, sodium chloride, dextrose, yeast extract, and pancreatic digest of casein with the purified water, and heat until solution is effected. Dissolve the sodium thioglycollate or thioglycolic acid in the solution and, if necessary, add 0 N sodium hydroxide so that, after sterilization, the solution will have a pH of 0 0. If filtration is necessary, heat the solution again without boiling, and filter while hot through moistened filter paper. Add the resazurin sodium solution, mix, and place the medium in suitable vessels that provide a ratio of surface to depth of medium such that not more than the upper half of the medium has undergone a color change indicative of oxygen uptake at the end of the incubation period. Sterilize using a validated process. If the medium is stored, store at a temperature between 0 and 0 in a sterile, airtight container. If more than the upper one-third of the medium has acquired a pink color, the medium may be restored once by heating the containers in a water-bath or in free-flowing steam until the pink color disappears and by cooling quickly, taking care to prevent the introduction of nonsterile air into the container. Fluid Thioglycollate Medium is to be incubated at 0 0 . Alternative Thioglycollate Medium Prepare a mixture having the same composition as that of the Fluid Thioglycollate Medium, but omitting the agar and the resazurin sodium solution, sterilize as directed above, and allow to cool prior to use. The pH after sterilization is 0 0. Incubate under anaerobic conditions for the duration of the incubation period. Alternative Fluid Thioglycollate Medium is to be incubated at 0 0 . SoybeanCasein Digest Medium Pancreatic Digest of Casein 0g
Papaic Digest of Soybean Meal 0 g Sodium Chloride Dibasic Potassium Phosphate Dextrose (C0H0O0H0O) 37 0g 0g 0/0 g

SoybeanCasein Digest Medium Purified Water 0 mL
Dissolve the solids in the Purified water, heating slightly to effect a solution. Cool the solution to room temperature, and adjust the pH with 0 N sodium hydroxide so that, after sterilization, it will have a pH of 0 0. Filter, if necessary to clarify, dispense into suitable containers, and sterilize using a validated procedure. Store at a temperature between 0 and 0 in a sterile well-closed container, unless it is intended for immediate use. SoybeanCasein Digest Medium is to be incubated at 0 0 . Media for Penicillins or Cephalosporins Where sterility test media are to be used in the Direct Inoculation of the Culture Medium method under Test for Sterility of the Product to be Examined, modify the preparation of Fluid Thioglycollate Medium and the SoybeanCasein Digest Medium as follows. To the containers of each medium, transfer aseptically a quantity of -lactamase sufficient to inactivate the amount of antibiotic in the specimen under test. Determine the quantity of lactamase required to inactivate the antibiotic by using a -lactamase preparation that has been assayed previously for its penicillin- or cephalosporin-inactivating power. [NOTE Supplemented -lactamase media can also be used in the membrane filtration test. ] Alternatively (in an area completely separate from that used for sterility testing), confirm that an appropriate amount of -lactamase is incorporated into the medium, following either method under Validation Test, using less than 0 colony-forming units (cfu) of Staphylococcus aureus (see Table 1) as the challenge. Typical microbial growth of the inoculated culture must be observed as a confirmation that the -lactamase concentration is appropriate. Suitability Tests The media used comply with the following tests, carried out before, or in parallel, with the test on the product to be examined.
STERILITY
38

Confirm the sterility of each sterilized batch of medium by incubating a portion of the media at the specified incubation temperature for 0 days. No growth of microorganisms occurs.
GROWTH PROMOTION TEST OF AEROBES, ANAEROBES, AND FUNGI
Test each lot of of ready-prepared medium and each batch of medium prepared either from dehydrated medium or from ingredients indicated in Table 1. Inoculate portions of Fluid Thioglycollate Medium with a small number (not more than 0 cfu) of the following microorganisms, using a separate portion of medium for each of the following species of microorganism: Clostridium sporogenes, Pseudomonas aeruginosa, and Staphylococcus aureus. Inoculate portions of Alternative Fluid Thioglycollate Inoculate
1
. Suitable strains of microorganisms are
Medium with a small number (not more than 0 cfu) of Clostridium sporogenes.
portions of SoybeanCasein Digest Medium with a small number (not more than 0 cfu) of the following microorganisms, using a separate portion of medium for each of the following species of microorganism: Aspergillus niger, Bacillus subtilis, and Candida albicans. Incubate for not more than 0 days in the case of bacteria and not more than 0 days in the case of fungi. The media are suitable if a clearly visible growth of the microorganisms occurs.
STORAGE
If prepared media are stored in unsealed containers, they can be used for 0 month, provided that they are tested for growth promotion within 0 weeks of the time of use and that color indicator requirements are met. If stored in tight containers, the media can be used for 0 year, provided that they are tested for growth promotion within 0 months of the time of use and that the color indicator requirements are met. Table 1. Strains of the Test Microorganisms Suitable for Use in the Growth Promotion Test and the Validation Test Aerobic bacteria
39

Staphylococcus aureus Bacillus subtilis Pseudomonas aeruginosa Anaerobic bacterium Clostridium sporogenes Fungi Candida albicans Aspergillus niger
0
3 2 1
ATCC 0, CIP 0, NCTC 0, NCIMB 0 ATCC 0, CIP 0, NCIMB 0 ATCC 0, NCIMB 0, CIP 0
ATCC 0, CIP 0, NCTC 0 or ATCC 0
ATCC 0, IP 0, NCPF 0 ATCC 0, IP 0, IMI 0
An alternative to Staphylococcus aureus is Bacillus subtilis (ATCC 0). An alternative microorganism is Micrococcus luteus (Kocuria rhizophila),
ATCC 0.
0
An alternative to Clostridium sporogenes, when a nonspore-forming
microorganism is desired, is Bacetroides vulgatus (ATCC 0). [NOTESeed-lot culture maintenance techniques (seed-lot systems) are used so that the viable microorganisms used for inoculation are not more than five passages removed from the original master seed lot.]
DILUTING AND RINSING FLUIDS FOR MEMBRANE FILTRATION Fluid A

PREPARATION
Dissolve 0 g of peptic digest of animal tissue in water to make 0 liter, filter or centrifuge to clarify, if necessary, and adjust to a pH of 0 0. Dispense into containers, and sterilize using a validated process.
PREPARATION FOR PENICILLINS OR CEPHALOSPORINS
40

Aseptically add to the above Preparation, if necessary, a quantity of sterile -lactamase sufficient to inactivate any residual antibiotic activity on the membranes after the solution of the test specimen has been filtered (see Media for Penicillins or Cephalosporins). Fluid D To each liter of Fluid A add 0 mL of polysorbate 0, adjust to a pH of 0 0, dispense into containers, and sterilize using a validated process. Use this fluid for articles containing lecithin or oil, or for devices labeled as sterile pathway. Fluid K Dissolve 0 g of peptic digest of animal tissue, 0 g of beef extract, and 0 g of polysorbate 0 in water to make 0 liter. Adjust the pH to obtain, after sterilization, a pH of 0 0. Dispense into containers, and sterilize using a validated process.
VALIDATION TEST Carry out a test as described below under Test for Sterility of the Product to be Examined using exactly the same methods, except for the following modifications. Membrane Filtration After transferring the content of the container or containers to be tested to the membrane, add an inoculum of a small number of viable microorganisms (not more than 0 cfu) to the final portion of sterile diluent used to rinse the filter. Direct Inoculation After transferring the contents of the container or containers to be tested (for catgut and other surgical sutures for veterinary use: strands) to the culture medium, add an inoculum of a small number of viable microorganisms (not more than 0 cfu) to the medium. In both cases use the same microorganisms as those described above under Growth Promotion Test of Aerobes, Anaerobes, and Fungi. Perform a growth promotion test as a positive control. Incubate all the containers containing medium for not more than 0 days.
41

If clearly visible growth of microorganisms is obtained after the incubation, visually comparable to that in the control vessel without product, either the product possesses no antimicrobial activity under the conditions of the test or such activity has been satisfactorily eliminated. The test for sterility may then be carried out without further modification. If clearly visible growth is not obtained in the presence of the product to be tested, visually comparable to that in the control vessels without product, the product possesses antimicrobial activity that has not been satisfactorily eliminated under the conditions of the test. Modify the conditions in order to eliminate the antimicrobial activity, and repeat the validation test. This validation is performed (a) when the test for sterility has to be carried out on a new product; and (b) whenever there is a change in the experimental conditions of the test. The validation may be performed simultaneously with the Test for Sterility of the Product to be Examined.
TEST FOR STERILITY OF THE PRODUCT TO BE EXAMINED Number of Articles to Be Tested Unless otherwise specified elsewhere in this chapter or in the individual monograph, test the number of articles specified in Table 3. If the contents of each article are of sufficient quantity (see Table 2), they may be divided so that equal appropriate portions are added to each of the specified media. [NOTEPerform sterility testing employing two or more of the specified media. ] If each article does not contain sufficient quantities for each medium, use twice the number of articles indicated in Table 3. Table 2. Minimum Quantity to be Used for Each Medium Minimum Quantity to be Used (unless otherwise justified and authorized)
Quantity per Container Liquids (other than anitbiotics) Less than 0 mL
The whole contents of each container
42

Minimum Quantity to be Used (unless otherwise justified and authorized) Half the contents of each container, but not less than 0 mL 0 mL
Quantity per Container 00 mL
Greater than 0 mL, and not greater than 0 mL Greater than 0 mL
0% of the contents of the container, but not less than 0 mL 0 mL
Antibiotic liquids Other preparations soluble in water or in isopropyl myristate Insoluble preparations, creams, and ointments to be suspended or emulsified Solids Less than 0 mg 0 mg or more, but less than 0 mg
The whole contents of each container to provide not less than 0 mg Use the contents of each container to provide not less than 0 mg
The whole contents of each container Half the contents of each container, but not less than 0 mg 0 mg 0 mg
0 mg0 g Greater than 0 g Devices Catgut and other surgical sutures for veterinary use Surgical dressing/cotton/gauze (in packages)
0 sections of a strand (each 0-cm long) 0 mg per package
43

Minimum Quantity to be Used (unless otherwise justified and authorized) The whole device
Quantity per Container Sutures and other individually packaged single-use material Other medical devices
The whole device, cut into pieces or disassembled
Table 3. Minimum Number of Articles to be Tested in Relation to the Number of Articles in the Batch Minimum Number of Items to be Tested for Each Medium (unless otherwise justified and authorized)*
Number of Items in the Batch Parenteral preparations Not more than 0 containers
0% or 0 containers, whichever is the greater 0 containers 0% or 0 containers, whichever is less 0% or 0 containers, whichever is less
More than 0 but not more than 0 containers More than 0 containers
For large-volume parenterals
Antibiotic solids Pharmacy bulk packages (<0 g) Pharmacy bulk packages (0 g) Bulks and blends Ophthalmic and other noninjectable preparations 44 0 containers 0 containers See Bulk solid products

Minimum Number of Items to be Tested for Each Medium (unless otherwise justified and authorized)* 0% or 0 containers, whichever is the greater 0 containers
Number of Items in the Batch Not more than 0 containers
More than 0 containers If the product is presented in the form of singledose containers, apply the scheme shown above for preparations for parenteral use. Devices
Catgut and other surgical sutures for veterinary use 0 % or 0 packages, whichever is the greater, up to a maximum total of 0 packages Not more than 0 articles 0% or 0 articles, whichever is greater 0 articles 0% or 0 articles, whichever is less Bulk solid products Up to 0 containers More than 0 containers, but not more than 0 containers More than 0 containers Each container 0% or 0 containers, whichever is greater 0% or 0 containers, whichever is greater
More than 0, but not more than 0 articles More than 0 articles
If the contents of one container are enough to inoculate the two media, this
45

Minimum Number of Items to be Tested for Each Medium (unless otherwise justified and authorized)*
Number of Items in the Batch
column gives the number of containers needed for both the media together. The test may be carried out using the technique of Membrane Filtration or by Direct Inoculation of the Culture Medium with the product to be examined. Appropriate negative controls are included. The technique of membrane filtration is used whenever the nature of the product permits; that is, for filterable aqueous preparations, for alcoholic or oily preparations, and for preparations miscible with, or soluble in, aqueous or oily solvents, provided these solvents do not have an antimicrobial effect in the conditions of the test. Membrane Filtration Use membrane filters having a nominal pore size not greater than 0 m whose effectiveness to retain microorganisms has been established. Cellulose nitrate filters, for example, are used for aqueous, oily, and weakly alcoholic solutions; and cellulose acetate filters, for example, are used for strongly alcoholic solutions. Specially adapted filters may be needed for certain products (e.g., for antibiotics). The technique described below assumes that membranes about 0 mm in diameter will be used. If filters of a different diameter are used, the volumes of the dilutions and the washings should be adjusted accordingly. The filtration apparatus and membrane are sterilized by appropriate means. The apparatus is designed so that the solution to be examined can be introduced and filtered under aseptic conditions: it permits the aseptic removal of the membrane for transfer to the medium, or it is suitable for carrying out the incubation after adding the medium to the apparatus itself.
AQUEOUS SOLUTIONS
If appropriate, transfer a small quantity of a suitable, sterile diluent such as Diluting and Rinsing Fluids for Membrane Filtration)
Fluid A (see
onto the membrane in the apparatus
and filter. The diluent may contain suitable neutralizing substances and/or appropriate inactivating substances, for example, in the case of antibiotics.
46

Transfer the contents of the container or containers to be tested to the membrane or membranes, if necessary, after diluting to the volume used in the Validation Test with the chosen sterile diluent, but using not less than the quantities of the product to be examined prescribed in Tables 2 and 3. Filter immediately. If the product has antimicrobial properties, wash the membrane not less than three times by filtering through it each time the volume of the chosen sterile diluent used in the Validation Test. Do not exceed a washing cycle of 0 times 0 mL, even if during validation it has been demonstrated that such a cycle does not fully eliminate the antimicrobial activity. Transfer the whole membrane to the culture medium or cut it aseptically into two equal parts, and transfer one half to each of two suitable media. Use the same volume of each medium as in the Validation Test. Alternatively, transfer the medium onto the membrane in the apparatus. Incubate the media for not less than 0 days.
SOLUBLE SOLIDS (OTHER THAN ANTIBIOTICS)
Use for each medium not less than the quantity prescribed in Tables 2 and 3 of the product dissolved in a suitable solvent, such as Membrane Filtration), Fluid A (Diluting and Rinsing Fluids for
and proceed with the test as described above for Aqueous
Solutions using a membrane appropriate to the chosen solvent.

OILS AND OILY SOLUTIONS
Use for each medium not less than the quantity of the product prescribed in Tables 2 and 3. Oils and oily solutions of sufficiently low viscosity may be filtered without dilution through a dry membrane. Viscous oils may be diluted as necessary with a suitable sterile diluent such as isopropyl myristate shown not to have antimicrobial activity in the conditions of the test. Allow the oil to penetrate the membrane by its own weight, and then filter, applying the pressure or suction gradually. Wash the membrane at least three times by filtering through it each time about 0 mL of a suitable sterile solution such as Diluting and Rinsing Fluids for Membrane Filtration) Fluid A (see
containing a suitable emulsifying
agent at a concentration shown to be appropriate in the validation of the test, for example polysorbate 0 at a concentration of 0 g per liter (Fluid K) . Transfer the membrane or
membranes to the culture medium or media, or vice versa, as described above for Aqueous Solutions, and incubate at the same temperatures and for the same times.
47

OINTMENTS AND CREAMS
Use for each medium not less than the quantities of the product prescribed in Tables 2 and 3. Ointments in a fatty base and emulsions of the water-in-oil type may be diluted to 0% in isopropyl myristate as described above, by heating, if necessary, to not more than 0 . In exceptional cases it may be necessary to heat to not more than 0 . Filter as rapidly as possible, and proceed as described above for Oils and Oily Solutions.
PREFILLED SYRINGES
For prefilled syringes without attached sterile needles, expel the contents of each syringe into one or two separate membrane filter funnels or into separate pooling vessels prior to transfer. If a separate sterile needle is attached, directly expel the syringe contents as indicated above, and proceed as directed for Aqueous Solutions. Test the sterility of the needle, using Direct Inoculation under Validation Test.
SOLIDS FOR INJECTION OTHER THAN ANTIBIOTICS
Constitute the test articles as directed on the label, and proceed as directed for Aqueous Solutions or Oils and Oily Solutions, whichever applies. [NOTEIf necessary, excess diluent can be added to aid in the constitution and filtration of the constituted test article. ]
ANTIBIOTIC SOLIDS FOR INJECTION
Pharmacy Bulk Packages, < 5 g From each of 0 containers, aseptically transfer about 0 mg of solids, into a sterile 0-mL conical flask, dissolve in about 0 mL of Fluid A (see Diluting and Rinsing Fluids for Membrane Filtration), and mix; or constitute, as directed in the labeling, each of 0 containers and transfer a quantity of liquid or suspension, equivalent to about 0 mg of solids, into a sterile 0-mL conical flask, dissolve in about 0 mL of Fluid A, and mix. Proceed as directed for Aqueous Solutions or Oils and Oily Solutions, whichever applies. Pharmacy Bulk Packages, 5 g From each of 0 containers, aseptically transfer about 0 g of solids into a sterile 0-mL conical flask, dissolve in about 0 mL of Fluid A, and mix; or constitute, as directed in the labeling, each of 0 containers and transfer a quantity of liquid, equivalent to about 0 g of solids, into a sterile 0-mL conical flask, dissolve in about 0 mL of Fluid A, and mix. Proceed as directed for Aqueous Solutions. 48

ANTIBIOTIC SOLIDS, BULKS, AND BLENDS
Aseptically remove a sufficient quantity of solids from the appropriate amount of containers (see Table 2), mix to obtain a composite, equivalent to about 0 g of solids, and transfer to a sterile 0-mL conical flask. Dissolve in about 0 mL of Fluid A, and mix. Proceed as directed for Aqueous Solutions.
STERILE AEROSOL PRODUCTS
For fluid products in pressurized aerosol form, freeze the containers in an alcohol-dry ice mixture at least at 0 for about 0 hour. If feasible, allow the propellant to escape before aseptically opening the container, and transfer the contents to a sterile pooling vessel. Add 0 mL of Fluid D to the pooling vessel, and mix gently. Proceed as directed for Aqueous Solutions or Oils and Oily Solutions, whichever applies.
DEVICES WITH PATHWAYS LABELED STERILE
Aseptically pass not less than 0 pathway volumes of Fluid D through each device tested. Collect the fluids in an appropriate sterile vessel, and proceed as directed for Aqueous Solutions or Oils and Oily Solutions, whichever applies. In the case of sterile, empty syringes, draw sterile diluent into the barrel through the sterile needle, if attached, or through a sterile needle attached for the purpose of the test, and express the contents into a sterile pooling vessel. Proceed as directed above. Direct Inoculation of the Culture Medium Transfer the quantity of the preparation to be examined prescribed in Tables 2 and 3 directly into the culture medium so that the volume of the product is not more than 0% of the volume of the medium, unless otherwise prescribed. If the product to be examined has antimicrobial activity, carry out the test after neutralizing this with a suitable neutralizing substance or by dilution in a sufficient quantity of culture medium. When it is necessary to use a large volume of the product, it may be preferable to use a concentrated culture medium prepared in such a way that it takes into account the subsequent dilution. Where appropriate, the concentrated medium may be added directly to the product in its container.
OILY LIQUIDS
49

Use media to which have been added a suitable emulsifying agent at a concentration shown to be appropriate in the validation of the test, for example polysorbate 0 at a concentration of 0 g per liter.
OINTMENTS AND CREAMS
Prepare by diluting to about 0 in 0 by emulsifying with the chosen emulsifying agent in a suitable sterile diluent such as Filtration). Fluid A (see Diluting and Rinsing Fluids for Membrane
Transfer the diluted product to a medium not containing an emulsifying agent.
Incubate the inoculated media for not less than 0 days. Observe the cultures several times during the incubation period. Shake cultures containing oily products gently each day. However, when thioglycollate medium or other similar medium is used for the detection of anaerobic microorganisms, keep shaking or mixing to a minimum in order to maintain anaerobic conditions.
CATGUT AND OTHER SURGICAL SUTURES FOR VETERINARIAN USE
Use for each medium not less than the quantities of the product prescribed in Tables 2 and 3. Open the sealed package using aseptic precautions, and remove three sections of the strand for each culture medium. Carry out the test on three sections, each 0-cm long, which have been cut off from the beginning, the center, and the end of the strand. Use whole strands from freshly opened cassette packs. Transfer each section of the strand to the selected medium. Use sufficient medium to cover adequately the material to be tested (0 mL to 0 mL).
SOLIDS
Transfer a quantity of the product in the form of a dry solid (or prepare a suspension of the product by adding sterile diluent to the immediate container), corresponding to not less than the quantity indicated in Tables 2 and 3. Transfer the material so obtained to 0 mL of Fluid Thioglycollate Medium, and mix. Similarly, transfer the same quantity to 0 mL of SoybeanCasein Digest Medium, and mix. Proceed as directed above.
PURIFIED COTTON, GAUZE, SURGICAL DRESSINGS, AND RELATED ARTICLES
From each package of cotton, rolled gauze bandage, or large surgical dressings being tested, aseptically remove two or more portions of 0- to 0-mg each from the innermost part 50

of the sample. From individually packaged, single-use materials, aseptically remove the entire article. Immerse the portions or article in each medium, and proceed as directed above.
STERILE DEVICES
Articles can be immersed intact or disassembled. To ensure that device pathways are also in contact with the media, immerse the appropriate number of units per medium in a volume of medium sufficient to immerse the device completely, and proceed as directed above. For extremely large devices, immerse those portions of the device that are to come into contact with the patient in a volume of medium sufficient to achieve complete immersion of those portions. For catheters where the inside lumen and outside are required to be sterile, either cut them into pieces such that the medium is in contact with the entire lumen or fill the lumen with medium, and then immerse the intact unit.
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 the material being tested renders the medium turbid so that the presence or absence of microbial growth cannot be readily determined by visual examination, 0 days after the beginning of incubation transfer portions (each not less than 0 mL) of the medium to fresh vessels of the same medium, and then incubate the original and transfer vessels for not less than 0 days. If no evidence of microbial growth is found, the product to be examined complies with the test for sterility. If evidence of microbial growth is found, the product to be examined does not comply with the test for sterility, unless it can be clearly demonstrated that the test was invalid for causes unrelated to the product to be examined. The test may be considered invalid only if one or more of the following conditions are fulfilled: a. The data of the microbiological monitoring of the sterility testing facility show a fault. b. A review of the testing procedure used during the test in question reveals a fault. c. Microbial growth is found in the negative controls. 51

d. After determination of the identity of the microorganisms isolated from the test, the growth of this species (or these species) may be ascribed unequivocally to faults with respect to the material and or the technique used in conducting the sterility test procedure. If the test is declared to be invalid, it is repeated with the same number of units as in the original test. If no evidence of microbial growth is found in the repeat test, the product examined complies with the test for sterility. If microbial growth is found in the repeat test, the product examined does not comply with the test for sterility.
APPLICATION OF THE TEST TO PARENTERAL PREPARATIONS, OPHTHALMIC, AND OTHER NONINJECTABLE PREPARATIONS REQUIRED TO COMPLY WITH THE TEST FOR STERILITY When using the technique of membrane filtration, use, whenever possible, the whole contents of the container, but not less than the quantities indicated in Tables 2 and 3, diluting where necessary to about 0 mL with a suitable sterile solution, such as (see Diluting and Rinsing Fluids for Membrane Filtration). When using the technique of direct inoculation of media, use the quantities shown in Tables 2 and 3, unless otherwise justified and authorized. The tests for bacterial and fungal sterility are carried out on the same sample of the product to be examined. When the volume or the quantity in a single container is insufficient to carry out the tests, the contents of two or more containers are used to inoculate the different media.
USP27
Fluid A
In appropriate cases, periodic testing of the different batches prepared from the same lot of
dehydrated medium is acceptable.
81 ANTIBIOTICSMICROBIAL ASSAYS The activity (potency) of antibiotics may be demonstrated under suitable conditions by their inhibitory effect on microorganisms. A reduction in antimicrobial activity also will reveal subtle changes not demonstrable by chemical methods. Accordingly, microbial or 52

biological assays remain generally the standard for resolving doubt with respect to possible loss of activity. This chapter summarizes these procedures for the antibiotics recognized in this Pharmacopeia for which microbiological assay remains the definitive method. Two general methods are employed, the cylinder-plate or plate assay and the turbidimetric or tube assay. The first depends upon diffusion of the antibiotic from a vertical cylinder through a solidified agar layer in a petri dish or plate to an extent such that growth of the added microorganism is prevented entirely in a circular area or zone around the cylinder containing a solution of the antibiotic. The turbidimetric method depends upon the inhibition of growth of a microbial culture in a uniform solution of the antibiotic in a fluid medium that is favorable to its rapid growth in the absence of the antibiotic.
APPARATUS All equipment is to be thoroughly cleaned before and after each use. Glassware for holding and transferring test organisms is sterilized by dry heat or by steam. Temperature Control Thermostatic control is required in several stages of a microbial assay, when culturing a microorganism and preparing its inoculum, and during incubation in plate and tube assays. Maintain the temperature of assay plates at 0 of the temperature selected. Closer control of the temperature (0 of the selected temperature) is imperative during incubation in a tube assay, and may be achieved in either circulated air or water, the greater heat capacity of water lending it some advantage over circulating air. Spectrophotometer Measuring transmittance within a fairly narrow frequency band requires a suitable spectrophotometer in which the wavelength of the light source can be varied or restricted by the use of a 0-nm filter or a 0-nm filter for reading the absorbance in a tube assay. For the latter purpose, the instrument may be arranged to accept the tube in which incubation takes place (see Turbidimetric Assay Receptacles), to accept a modified cell fitted with a drain that facilitates rapid change of content, or preferably, fixed with a flow-through cell for 53

a continuous flow-through analysis; set the instrument at zero absorbance with clear, uninoculated broth prepared as specified for the particular antibiotic, including the same amount of test solution and formaldehyde as found in each sample. NOTEEither absorbance or transmittance measurement may be used for preparing inocula. Cylinder-Plate Assay Receptacles For assay plates, use glass or plastic petri dishes (approximately 0 0 mm) having covers of suitable material. For assay cylinders, use stainless steel or porcelain cylinders with the following dimensions, each dimension having a tolerance of 0 mm: outside diameter 0 mm; inside diameter 0 mm; and length 0 mm. Carefully clean cylinders to remove all residues. An occasional acid bath, e.g., with about 0 N nitric acid or with chromic acid (see Cleaning Glass Apparatus 1051) is needed. Turbidimetric Assay Receptacles For assay tubes, use glass or plastic test tubes, e.g., 0 0 mm or 0 0 mm that are relatively uniform in length, diameter, and thickness and substantially free from surface blemishes and scratches. Tubes that are to be placed in the spectrophotometer are matched and are without scratches or blemishes. Cleanse thoroughly to remove all antibiotic residues and traces of cleaning solution, and sterilize tubes that have been used previously, before subsequent use.
MEDIA AND DILUENTS Media The media required for the preparation of test organism inocula are made from the ingredients listed herein. Minor modifications of the individual ingredients, or reconstituted dehydrated media, may be substituted, provided the resulting media possess equal or better growth-promoting properties and give a similar standard curve response. Dissolve the ingredients in water to make 0 liter, and adjust the solutions with either 0 N sodium hydroxide or 0 N hydrochloric acid as required, so that after steam sterilization the pH is as specified. 54

MEDIUM 1 Peptone 0g
Pancreatic Digest of Casein 0 g Yeast Extract Beef Extract Dextrose Agar Water 0g 0g 0g 0g 0 mL
pH after sterilization: 0 0. MEDIUM 2 Peptone Yeast Extract Beef Extract Agar Water 0g 0g 0g 0g 0 mL
pH after sterilization: 0 0. MEDIUM 3 Peptone Yeast Extract Beef Extract Sodium Chloride Dextrose 0g 0g 0g 0g 0g
55

Dibasic Potassium Phosphate 0g
Monobasic Potassium Phosphate 0 g Water pH after sterilization: 0 0.

MEDIUM 4
0 mL
Same as Medium 0, except for the additional ingredient 0 g of Dextrose.

MEDIUM 5
Same as Medium 0, except that the final pH after sterilization is 0 0.

MEDIUM 8
Same as Medium 0, except that the final pH after sterilization is 0 0. MEDIUM 9 Pancreatic Digest of Casein Papaic Digest of Soybean Sodium Chloride 0g 0g 0g
Dibasic Potassium Phosphate 0 g Dextrose Agar Water pH after sterilization: 0 0.

MEDIUM 10
0g 0g 0 mL
Same as Medium 0, except to use 0 g of Agar instead of 0 g, and to add 0 mL of Polysorbate 0 after boiling the medium to dissolve the agar. pH after sterilization: 0 0.
56

MEDIUM 11
Same as Medium 0, except that the final pH after sterilization is 0 0. MEDIUM 13 Dextrose Peptone Water pH after sterilization: 0 0. MEDIUM 19 Peptone Yeast Extract Beef Extract Sodium Chloride Dextrose Agar Water 0g 0g 0g 0g 0g 0g 0 mL 0g 0g 0 mL
pH after sterilization: 0 0.
MEDIUM 32
Same as Medium 0, except for the additional ingredient 0 g of Manganese Sulfate. MEDIUM 34 Glycerol Peptone Beef Extract 0g 0g 0g
57

Sodium Chloride Water 0g 0 mL
MEDIUM 35
Same as Medium 0, except for the additional ingredient 0 g of Agar. MEDIUM 36 Pancreatic Digest of Casein 0 g Papaic Digest of Soybean Sodium Chloride Agar Water 0g 0g 0g 0 mL
MEDIUM 39
Same as Medium 3, except that the final pH after sterilization is 0 0. MEDIUM 40 Yeast Extract Polypeptone Dextrose 0g 0g 0g
Monobasic Potassium Phosphate 0 g Polysorbate 0 Agar Water 0g 0g 0 mL
58

pH after sterilization: 0 0. MEDIUM 41 Pancreatic Digest of Casein Dextrose Yeast Extract Sodium Citrate 0g 0g 0g 0g
Monobasic Potassium Phosphate 0 g Dibasic Potassium Phosphate Water pH after sterilization: 0 0. Phosphate Buffers and Other Solutions Prepare as follows, or by other suitable means, the potassium phosphate buffers required for the antibiotic under assay. The buffers are sterilized after preparation, and the pH specified in each case is the pH after sterilization. BUFFER NO. 1, 1 PERCENT, PH 6.0 Dissolve 0 g of dibasic potassium phosphate and 0 g of monobasic potassium phosphate in 0 mL of water. Adjust with 0 N phosphoric acid or 0 N potassium hydroxide to 0 0. BUFFER NO. 3, 0.1 M, PH 8.0 Dissolve 0 g of dibasic potassium phosphate and 0 g of monobasic potassium phosphate in 0 mL of water. Adjust the pH with 0 N phosphoric acid or 0 N potassium hydroxide to 0 0. BUFFER NO. 4, 0.1 M, PH 4.5 Dissolve 0 g of monobasic potassium phosphate in 0 mL of water. Adjust the pH with 0 N phosphoric acid or 0 N potassium hydroxide to 0 0. BUFFER NO. 6, 10 PERCENT, PH 6.0 Dissolve 0 g of dibasic potassium phosphate and 0 g of monobasic potassium phosphate in 0 mL of water. Adjust the pH with 0 N phosphoric acid or 0 N potassium hydroxide to 0 0. 0g 0 mL
59

BUFFER NO. 10, 0.2 M, PH 10.5 Dissolve 0 g of dibasic potassium phosphate in 0 mL of water, and add 0 mL of 0 N potassium hydroxide. Adjust the pH with 0 N phosphoric acid or 0 N potassium hydroxide to 0 0. BUFFER NO. 16, 0.1 M, PH 7.0 Dissolve 0 g of dibasic potassium phosphate and 0 g of monobasic potassium phosphate in 0 mL of water. Adjust with 0 N phosphoric acid or 0 N potassium hydroxide to a pH of 0 0. OTHER SOLUTIONS Use the substances specified under Reagents, Indicators, and Solutions. For water, use Purified Water. For saline, use Sodium Chloride Injection. Dilute formaldehyde is Formaldehyde Solution diluted with water 0:0.
UNITS AND REFERENCE STANDARDS The potency of antibiotics is designated in either Units or g of activity. In each case the Unit or g of antibiotic activity is established and defined by the designated federal master standard for that antibiotic. The corresponding USP Reference Standard is calibrated in terms of the master standard. USP Reference Standards for antibiotic substances are held and distributed by the U.S. Pharmacopeial Convention, Inc. The concept of g of activity originated from the situation where the antibiotic preparation selected as the reference standard was thought to consist entirely of a single chemical entity and was therefore assigned a potency of 0 g per mg. In several such instances, as a result of the development of manufacturing and purification methods for particular antibiotics, preparations became available that contained more than 0 g of activity per mg. It was then understood that such preparations had an activity equivalent to a given number of g of the original reference standard. In most instances, however, the g of activity is exactly equivalent numerically to the g (weight) of the pure substance. Complications arise in some situations, e.g., where an antibiotic exists as the free base and in salt form, and the g of activity has been defined in terms of one such form; where the antibiotic substance consists of a number of components having close chemical similarity but differing antibiotic activity; or where the potencies of a family of antibiotics are expressed in terms of a reference standard consisting of a single member which, however, might itself be heterogeneous. In such cases the g of activity defined in terms of a
60

Master Standard is tantamount to a Unit. The g of activity should therefore not be assumed necessarily to correspond to the g (weight) of the antibiotic substance.
PREPARATION OF THE STANDARD To prepare a stock solution, dissolve a quantity of the USP Reference Standard of a given antibiotic, accurately weighed, or the entire contents of a vial of USP Reference Standard, where appropriate, in the solvent specified in that table, and then dilute to the required concentration as indicated. Store in a refrigerator, and use within the period indicated. On the day of the assay, prepare from the stock solution 0 or more test dilutions, the successive solutions increasing stepwise in concentration, usually in the ratio of 0:0 for a cylinder-plate assay or smaller for a turbidimetric assay. Use the final diluent specified and a sequence such that the middle or median has the concentration designated.
PREPARATION OF THE SAMPLE From the information available for the preparation to be assayed (the Unknown), assign to it an assumed potency per unit weight or volume, and on this assumption prepare on the day of the assay a stock solution and test dilution as specified for each antibiotic but with the same final diluent as used for the USP Reference Standard. The assay with 0 levels of the Standard requires only one level of the Unknown at a concentration assumed equal to the median level of the Standard.
ORGANISMS AND INOCULUM Test Organisms The test organism for each antibiotic is listed in Table 2, together with its identification number in the American Type Culture Collection. The method of assay is given for each in Table 1. Maintain a culture on slants of the medium and under the incubation conditions specified in Table 3, and transfer weekly to fresh slants. For K. pneumoniae use a noncapsulated culture. For Enterococcus hirae, stab cultures may be used. Table 1. Preparation of Stock Solutions and Test Dilutions of Reference Standards
61

Stock Solution Test Dilution Media n Dose (g of activit y or Units Final per Diluent mL) Water 0 g
Antibiotic and Type Initial Solvent (and of initial Assay [Cylinderconcentration Final Stock plate where specified); Concentratio Use (CP) or Further Diluent, if n Withi Turbidimetric (T)] different per mL n Amikacin (T) Water 0 mg 0 days Same day Same day 0 days Same day 0 days 0 days 0 days 0 days 0 days
Amphotericin B (CP) Dimethyl sulfoxide
0 mg
B. 0
0 g
Bacitracin Zinc (CP)
0 N hydrochloric acid B. 0
0U
B. 0
0U
Bleomycin (CP)
0U
B. 0
0U
Candicidin (T)
Dimethyl sulfoxide
0 mg
Water
0 g
Capreomycin (T)
Water
0 mg
Water
0 g
Carbenicillin (CP)
B. 0
0 mg
B. 0
0 g
Cephalothin (CP)
B. 0
0 mg
B. 0
0 g
Cephapirin (CP)
B. 0
0 mg
B. 0
0 g
Chloramphenicol (T)
Alcohol (0 mg/mL); [Water]
0 mg
Water
0 g
62

Antibiotic and Type Initial Solvent (and of initial Assay [Cylinderconcentration Final Stock plate where specified); Concentratio Use (CP) or Further Diluent, if n Withi Turbidimetric (T)] different per mL n Chlortetracycline (T) Cloxacillin (CP) 0 N hydrochloric acid B. 0 0 mg 0 days 0 days Same day 0 days 0 days 0 days 0 days 0 days 0 days 0 days
0 mg
B. 0
0 g
Colistimethate Sodium (CP) Colistin (CP)
Water (0 mg/mL); [ B. 0] Water (0 mg/mL); [ B. 0] Water
0 mg
B. 0
0 g
0 mg
B. 0
0 g
Cycloserine (T)
0 mg
Water
0 g
Demeclocycline (T)
0 N hydrochloric acid B. 0
0 mg
Water
0 g
Dihydrostreptomyci n (CP) Dihydrostreptomyci n (T) Doxycycline (T)
0 mg
B. 0
0 g
Water
0 mg
Water
0 g
0 N hydrochloric acid Methanol (0 mg/mL); [ B. 0]
0 mg
Water
0 g
Erythromycin (CP)
0 mg
B. 0
0 g
63

Stock Solution Test Dilution Media n Dose (g of activit y or Units Final per Diluent mL) B. 0 0 g
Antibiotic and Type Initial Solvent (and of initial Assay [Cylinderconcentration Final Stock plate where specified); Concentratio Use (CP) or Further Diluent, if n Withi Turbidimetric (T)] different per mL n Gentamicin (CP) B. 0 0 mg 0 days 0 days 0 days 0 days 0 days Same day 0 days 0 days 0 days 0 days
Gramicidin (T)
Alcohol 0%
0 mg
Alcohol 0% Water
0 g
Kanamycin (T)
Water
0 mg
0 g
Methacycline (T)
Water
0 mg
Water
0 g
Nafcillin (CP)
B. 0
0 mg
B. 0
0 g
Natamycin (CP)
Dimethyl sulfoxide
0 mg
B. 0
0 g
Neomycin (CP)
B. 0
0 mg
B. 0
0 g
Neomycin (T)
B. 0
0 g
B. 0
0 g
Netilmicin (CP)
B. 0
0 mg
B. 0
0 g
Novobiocin (CP)
Alcohol (0 mg/mL); [ B. 0]
0 mg
B. 0
0 g
64

Stock Solution Test Dilution Media n Dose (g of activit y or Units Final per Diluent mL) B. 0 0U
Antibiotic and Type Initial Solvent (and of initial Assay [Cylinderconcentration Final Stock plate where specified); Concentratio Use (CP) or Further Diluent, if n Withi Turbidimetric (T)] different per mL n Nystatin (CP) Dimethylformamid e 0 N hydrochloric acid B. 0 0,0 U Same day 0 days 0 days 0 days 0 days 0 day 0 days 0 days 0 day
Oxytetracycline (T)
0 mg
Water
0 g
Paromomycin (CP)
0 mg
B. 0
0 g
Penicillin G (CP)
B. 0
0,0 U
B. 0
0U
Polymyxin B (CP)
Water; [ B. 0]
0,0 U
B. 0
0U
Rolitetracycline (T) Sisomicin (CP)
Water B. 0
0 mg 0 mg
Water B. 0
0 g 0 g
Streptomycin (T)
Water
0 mg
Water
0 g
Tetracycline (T)
0 N hydrochloric acid Dimethyl sulfoxide
0 mg
Water
0 g
Thiostrepton (T)
0U
Same Dimethyl day sulfoxide 0 day B. 0
0U
Ticarcillin (CP)
B. 0
0 mg
0 g
65

Antibiotic and Type Initial Solvent (and of initial Assay [Cylinderconcentration Final Stock plate where specified); Concentratio Use (CP) or Further Diluent, if n Withi Turbidimetric (T)] different per mL n Tobramycin (T) Water 0 mg 0 days Same day
Troleandomycin (T)
Isopropyl alcoholwater (0:0) Methanol (0 mg/mL); [ B. 0]
0 mg
Water
0 g
Tylosin (T)
0 mg
0 B.0: days methano l (0:0) 0 days B. 0
0 g
Vancomycin (CP)
Water
0 mg
0 g
NOTESB denotes buffer, and the number following refers to the potassium phosphate buffers defined in this chapter. For amphotericin B, colistimethate sodium, and nystatin, prepare the USP Reference Standard solutions and the sample test solution simultaneously. For amphotericin B, further dilute the stock solution with dimethyl sulfoxide to give concentrations of 0, 0, 0, 0, and 0 g per mL prior to making the test dilutions. The Test Dilution of the sample should contain the same amount of dimethyl sulfoxide as the test dilutions of the USP Reference Standard. For bacitracin zinc, each of the Standard test dilutions should contain the same amount of hydrochloric acid as the Test Dilution of the sample. For neomycin turbidimetric assay, dilute the 0-g-per-mL stock solution quantitatively with Buffer No. 0 to obtain a solution having a concentration equivalent to 0 g of neomycin per mL. To separate 0-mL volumetric flasks add 0, 0, 0, 0, and 0 mL of this solution, add 0 mL of 0 N hydrochloric acid to 66

Stock Solution Test Dilution Media n Dose (g of activit y or Units Final per Diluent mL)
Antibiotic and Type Initial Solvent (and of initial Assay [Cylinderconcentration Final Stock plate where specified); Concentratio Use (CP) or Further Diluent, if n Withi Turbidimetric (T)] different per mL n
each flask, dilute with Buffer No. 0 to volume, and mix to obtain solutions having concentrations of 0, 0, 0, 0, and 0 g of neomycin per mL. Use these solutions to prepare the standard response line. For nystatin, further dilute the stock solution with dimethylformamide to give concentrations of 0, 0, 0, 0, and 0 Units per mL prior to making the test dilutions. Prepare the standard response line solutions simultaneously with dilutions of the sample to be tested. The Test Dilution of the sample should contain the same amount of dimethylformamide as the test dilutions of the Standard. Use red low-actinic glassware. For Polymyxin B, prepare the stock solution by adding 0 mL of water for each 0 mg of the weighed USP Reference Standard material. Table 2. Test Organisms for Antibiotics Assayed by the Procedure Indicated in Table 1 Antibiotic Amikacin Amphotericin B Bacitracin Bleomycin Candicidin Capreomycin Test Organism Staphylococcus aureus Saccharomyces cerevisiae Micrococcus luteus Mycobacterium smegmatis Saccharomyces cerevisiae Klebsiella pneumoniae ATCC
*
Number 0 0 0 0 0 0
67

Antibiotic Carbenicillin Cephalothin Cephapirin Chloramphenicol Chlortetracycline Cloxacillin Colistimethate Sodium Colistin Cycloserine Demeclocycline Dihydrostreptomycin (CP) Dihydrostreptomycin (T) Doxycycline Erythromycin Gentamicin Gramicidin Kanamycin Test Organism Pseudomonas aeruginosa Staphylococcus aureus Staphylococcus aureus ATCC
*
Number 0 0 0
Escherichia coli
Staphylococcus aureus Staphylococcus aureus
0 0
Bordetella bronchiseptica Bordetella bronchiseptica Staphylococcus aureus Staphylococcus aureus
0 0 0 0
Bacillus subtilis
Klebsiella pneumoniae Staphylococcus aureus Micrococcus luteus Staphylococcus epidermidis Enterococcus hirae Staphylococcus aureus
0 0 0 0 0 0
68

Antibiotic Methacycline Nafcillin Neomycin (CP) Neomycin (T) Netilmicin Novobiocin Nystatin Oxytetracycline Paromomycin Penicillin G Polymyxin B Rolitetracycline Sisomicin Spectinomycin Streptomycin (T) Tetracycline Thiostrepton (T) Tobramycin Troleandomycin Tylosin Test Organism Staphylococcus aureus Staphylococcus aureus Staphylococcus epidermidis Klebsiella pneumoniae Staphylococcus epidermidis Staphylococcus epidermidis Saccharomyces cerevisiae Staphylococcus aureus Staphylococcus epidermidis Staphylococcus aureus Bordetella bronchiseptica Staphylococcus aureus Staphylococcus epidermidis Escherichia coli ATCC
*
Number 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Klebsiella pneumoniae Staphylococcus aureus Enterococcus hirae Staphylococcus aureus Klebsiella pneumoniae Staphylococcus aureus 69
0 0 0 0 0 0

Antibiotic Vancomycin
*
Test Organism Bacillus subtilis
ATCC
Number 0
American Type Culture Collection, 0 University Boulevard, Manassas VA 0-0. (http://www.atcc.org). Table 3. Preparation of Inoculum Suggested Inoculum Composition
Incubation Conditions
Test Organism Amount Temp. & (mL per (ATCC No.) Medium ( ) Time Medium 0 mL) Bacillus subtilis (0) 0 0 to 0 0 days 0
Antibiotics Assayed
As Dihydrostreptomycin required As Vancomycin required 0 Colistimethate Sodium, Colistin, Polymyxin B Chloramphenicol
Bordetella bronchiseptica (0) Escherichia coli (0) Klebsiella pneumoniae (0)
0 to 0 0 hr.
0 to 0 0 hr.
0 to 0 0 to 0 hr.
Capreomycin
Streptomycin, Troleandomycin, Dihydrostreptomycin Neomycin
70

Incubation Conditions Suggested Inoculum Composition
Test Organism Amount Temp. & (mL per (ATCC No.) Medium ( ) Time Medium 0 mL) Micrococcus luteus (0) Micrococcus luteus (0) Mycobacterium smegmatis (0) Pseudomonas aeruginosa (0) Saccharomyces cerevisiae (0) 0 0 to 0 0 hr. 0 0 to 0 0 hr.
Antibiotics Assayed
0 0
0 0
Erythromycin Bacitracin
0 to 0 0 hr.
Bleomycin
0 to 0 0 hr.
Carbenicillin
0 to 0 0 hr.
Candicidin
0 Saccharomyces cerevisiae (0) Staphylococcus aureus (0) Staphylococcus aureus (0) 0 0 to 0 0 hr. 0
0 0
Amphotericin B Nystatin
0 to 0 0 to 0 hr.
0-0
Tylosin
0 to 0 0 hr.
Cephalothin, Cephapirin, Cloxacillin
71

Test Organism Amount Temp. & (mL per (ATCC No.) Medium ( ) Time Medium 0 mL) 0 0 0 0 0 0
Antibiotics Assayed Nafcillin Penicillin G Amikacin, Chlortetracycline, Demeclocycline, Doxycycline, Methacycline, Oxytetracycline, Rolitetracycline, Tetracycline Kanamycin Cycloserine Tobramycin Netilmicin
0 0 0 Staphylococcus epidermidis (0) 0 0 to 0 0 hr. 0
0 0 0 0
0 0 0 0
0 0 0 0 0
Novobiocin Gentamicin, Sisomicin Neomycin Paromomycin Gramicidin
Enterococcus hirae (0)
0 to 0 0 to 0 hr.
72

Test Organism Amount Temp. & (mL per (ATCC No.) Medium ( ) Time Medium 0 mL) 0 0 to 0 0 to 0 hr. 0 0
Antibiotics Assayed Thiostrepton
NOTE For Pseudomonas aeruginosa (ATCC 0) in the assay of Carbenicillin, use 0 mL of a 0:0 dilution of the stock suspension per 0 mL of Medium 10. Preparation of Inoculum Preparatory to an assay, remove the growth from a recently grown slant or culture of the organism, with 0 mL of sterile saline TS and sterile glass beads. Inoculate the surface of 0 mL of the agar medium specified for that organism in Table 3 and contained on the flat side of a Roux bottle except in the case of Enterococcus hirae and Staphylococcus aureus (ATCC 0), which are grown in a liquid medium. Spread the suspension evenly over the surface of the agar with the aid of sterile glass beads, and incubate at the temperature shown for approximately the indicated length of time. At the end of this period, prepare the stock suspension by collecting the surface growth in 0 mL of sterile saline TS, except for Bleomycin (use 0 mL of Medium 34). Determine by trial the quantity of stock suspension to be used as the Inoculum, starting with the volume suggested in Table 3. The trial tests should be incubated for the times indicated in the section Turbidimetric Method under Procedure. Adjust the quantity of Inoculum on a daily basis, if necessary, to obtain the optimum dose-response relationship from the amount of growth of the test organism in the assay tubes and the length of the time of incubation. At the completion of the incubation periods described in the section Turbidimetric Method under Procedure, tubes containing the median dose of the Standard should have absorbances of at least 0 absorbance unit, except for Amikacin, Chlortetracycline, Gramicidin, and Tetracycline (0 absorbance unit), and Capreomycin, Methacycline, and Tobramycin (0 absorbance unit). 73

For the cylinder-plate assay, determine by trial the proportions of stock suspension to be incorporated in the Inoculum, starting with the volumes indicated in Table 3, that result in satisfactory demarcation of the zones of inhibition of about 0 to 0 mm in diameter and giving a reproducible dose relationship. Prepare the inoculum by adding a portion of stock suspension to a sufficient amount of agar medium that has been melted and cooled to 0 to 0 , and swirling to attain a homogeneous suspension.
PROCEDURE Assay Designs Microbial assays gain markedly in precision by the segregation of relatively large sources of potential error and bias through suitable experimental designs. In a cylinder-plate assay, the essential comparisons are restricted to relationships between zone diameter measurements within plates, exclusive of the variation between plates in their preparation and subsequent handling. To conduct a turbidimetric assay so that the differences in observed turbidity will reflect the differences in the antibiotic concentration requires both greater uniformity in the environment created for the tubes through closer thermostatic control of the incubator and the avoidance of systematic bias by use of a random placement of replicate tubes in separate tube racks, each rack containing one complete set of treatments. The essential comparisons are then restricted to relationships between the observed turbidities within racks. NOTEFor some purposes, the practice is to design the assay so that a set of treatments consists of not fewer than three tubes for each sample and standard concentration, and each set is placed in a single rack. Within these restrictions, the assay design recommended is a 0-level assay with a standard curve. For this assay with a standard curve, prepare solutions of 0, 0, or more test dilutions, provided they include one corresponding to the reference concentration ( S
0),
of the Standard and a solution of a single median test level of the Unknown as
described under Preparation of Standard and Preparation of the Sample. Consider an assay as preliminary if its computed potency with either design is less than 0% or more than 0% of that assumed in preparing the stock solution of the Unknown. In such a case, adjust its assumed potency accordingly and repeat the assay. 74

Microbial determinations of potency are subject to inter-assay as well as intra-assay variables, so that two or more independent assays are required for a reliable estimate of the potency of a given assay preparation or Unknown. Starting with separately prepared stock solutions and test dilutions of both the Standard and the Unknown, repeat the assay of a given Unknown on a different day. If the estimated potency of the second assay differs significantly, as indicated by the calculated standard error, from that of the first, conduct one or more additional assays. The combined result of a series of smaller, independent assays spread over a number of days is a more reliable estimate of potency than that from a single large assay with the same total number of plates or tubes. Cylinder-Plate Method To prepare assay plates using Petri dishes, place 0 mL of Medium 0 in each of the required number of plates, and allow it to harden into a smooth base layer of uniform depth, except for Amphotericin B and Nystatin, where no separate base layer is used. For Erythromycin, Gentamicin, Neomycin B, Paromomycin, and Sisomicin, use Medium 11. For Bleomycin, use 0 mL of Medium 35. For Dihydrostreptomycin use Medium 5. For Vancomycin, use 0 mL of Medium 8. For Carbenicillin, Colistimethate Sodium, Colistin, and Polymyxin B, use Medium 9. For Netilmicin, use 0 mL of Medium 11. Add 0 mL of seed layer inoculum (see Preparation of Inoculum and Table 3), prepared as directed for the given antibiotic, except for Bleomycin (use 0 mL), for Netilmicin (use 0 mL), and for Nystatin and Amphotericin B (use 0 mL), tilting the plate back and forth to spread the inoculum evenly over the surface, and allow it to harden. Drop 0 assay cylinders on the inoculated surface from a height of 0 mm, using a mechanical guide or other device to insure even spacing on a radius of 0 cm, and cover the plates to avoid contamination. After filling the 0 cylinders on each plate with dilutions of antibiotic containing the test levels specified below, incubate the plates at 0 to 0 , or at the temperature specified below for the individual case, for 0 to 0 hours, remove the cylinders, and measure and record the diameter of each zone of growth inhibition to the nearest 0 mm. Incubate the plates at 0 to 0 for Amphotericin B and Nystatin. Incubate at 0 to 0 for Novobiocin. Incubate at 0 to 0 for Carbenicillin, Colistimethate Sodium, Colistin, Dihydrostreptomycin, Gentamicin, Neomycin, Netilmicin, Paromomycin, Polymyxin B, Sisomicin, and Vancomycin.
75

For the 0-level assay with a standard curve, prepare dilutions representing 0 test levels of the Standard ( S 0 to S 0) and a single test level of the Unknown U 0 corresponding to S 0 of the standard curve, as defined under Preparation of the Standard and Preparation of the Sample. For deriving the standard curve, fill alternate cylinders on each of 0 plates with the median test dilution ( S 0) of the Standard and each of the remaining 0 cylinders with one of the other four dilutions of the Standard. Repeat the process for the three dilutions of the Standard. For each Unknown, fill alternate cylinders on each of 0 plates with the median test dilution of the Standard ( S 0), and the remaining 0 cylinders with the corresponding test dilution ( U 0) of the Unknown. Turbidimetric Method On the day of the assay, prepare the necessary doses by dilution of stock solutions of the Standard and of each Unknown as defined under Preparation of the Standard and Preparation of the Sample. Add 0 mL of each dose, except for Gramicidin, Thiostrepton and Tylosin (use 0 mL) to each of 0 prepared test tubes, and place the 0 replicate tubes in a position, selected at random, in a test tube rack or other carrier. Include similarly in each rack 0 or 0 control tubes containing 0 mL of the test diluent (see Table 1) but no antibiotic. Upon completion of the rack of test solutions (with candicidin within 0 minutes of the time when water is added to the methyl sulfoxide stock solution), add 0 mL of inoculum to each tube in the rack in turn, and place the completed rack immediately in an incubator or a water bath maintained at 0 to 0 , except for Candicidin (incubate at 0 to 0 ). Incubate the tubes for 0 to 0 hours, except for Capreomycin, Chloramphenicol, Cycloserine, Dihydrostreptomycin, Spectinomycin, Streptomycin, and Troleandomycin (incubate these for 0 to 0 hours), Tylosin (incubate for 0 to 0 hours), and Candicidin (incubate for 0 to 0 hours). After incubation add 0 mL of dilute formaldehyde to each tube, except for tylosin (heat the rack in a water bath at 0 to 0 for 0 to 0 minutes or in a steam bath for 0 to 0 minutes, and bring to room temperature), taking one rack at a time, and read its transmittance or absorbance in a suitable spectrophotometer fitted with a 0-nm or 0-nm filter (see Spectrophotometer under Apparatus). For the 0-level assay with a standard curve, prepare dilutions representing 0 test levels of the Standard ( S 0 to S 0) and a single test level ( U 0) of each of up to 0 Unknowns corresponding to S 0 of the Standard. Prepare also an extra S 0 as a test of growth. Add 0 mL of each test dilution, except for Gramicidin, Thiostrepton, and Tylosin (use 0 mL) to 0 76

tubes and 0 mL of antibiotic-free diluent to 0 tubes as controls. Distribute one complete set, including 0 tubes of controls, to a tube rack, intermingling them at random. Add 0 mL of inoculum, except for Thiostrepton (use 0 mL of inoculum), incubate, add 0 mL of dilute formaldehyde, and complete the assay as directed above. Determine the exact duration of incubation by observation of growth in the reference concentration (median dose) of the dilutions of the standard ( S 0).
CALCULATION To calculate the potency from the data obtained either by the cylinder-plate or by the turbidimetric method, proceed in each case as directed under Potencies Interpolated from a Standard Curve (see Design and Analysis of Biological Assays 111), using a log transformation, straight-line method with a least squares fitting procedure, and a test for linearity. Where a number of assays of the same material are made with the same standard curve, calculate the coefficient of variation of results of all of the assays of the material. Where more than one assay is made of the same material with different standard curves, average the two or more values of the potency.
85 BACTERIAL ENDOTOXINS TEST Portions of this general chapter have been harmonized with the corresponding texts of the European Pharmacopeia and/or the Japanese Pharmacopeia. Those portions that are not harmonized are marked with symbols ( ) to specify this fact.
This chapter provides a test to detect or quantify bacterial endotoxins that may be present in or on the sample of the article(s) to which the test is applied. It uses Limulus Amebocyte Lysate (LAL) obtained from the aqueous extracts of circulating amebocytes of horseshoe crab (Limulus polyphemus or Tachypleus tridentatus) which has been prepared and characterized for use as an LAL Reagent.1 There are two types of techniques for this test: the gel-clot techniques, which are based on gel formation, and the photometric techniques. The latter include a turbidimetric method, which is based on the development of turbidity after cleavage of an endogenous substrate, 77

and a chromogenic method, which is based on the development of color after cleavage of a synthetic peptide-chromogen complex. Proceed by any one of these techniques, unless otherwise indicated in the monograph. In case of dispute, the final decision is based on the gel-clot techniques, unless otherwise indicated in the monograph. In the gel-clot techniques, the reaction endpoint is determined from dilutions of the material under test in direct comparison with parallel dilutions of a reference endotoxin, and quantities of endotoxin are expressed in USP Endotoxin Units (USP-EU). [NOTEOne USP-EU is equal to one IU of endotoxin. ] Since LAL Reagents have been formulated to be used also for turbidimetric or colorimetric tests, such tests may be used to comply with the requirements. These tests require the establishment of a standard regression curve; the endotoxin content of the test material is determined by interpolation from the curve. The procedures include incubation for a preselected time of reacting endotoxin and control solutions with LAL Reagent and reading of the spectrophotometric light absorbance at suitable wavelengths. In the endpoint turbidimetric procedure the reading is made immediately at the end of the incubation period. In the endpoint colorimetric procedure the reaction is arrested at the end of the preselected time by the addition of an enzyme reaction-terminating agent prior to the readings. In the turbidimetric and colorimetric kinetic assays the absorbance is measured throughout the reaction period and rate values are determined from those readings.
APPARATUS AND GLASSWARE Depyrogenate all glassware and other heat-stable materials in a hot-air oven using a validated process.2 Commonly used minimum time and temperature settings are 0 minutes at 0 . If employing plastic apparatus, such as microplates and pipet tips for automatic pipetters, use only that which has been shown to be free of detectable endotoxin and not to interfere with the test. [NOTEIn this chapter, the term tube includes any other receptacle such as a micro-titer well. ]
PREPARATION OF THE STANDARD ENDOTOXIN STOCK SOLUTION AND STANDARD SOLUTIONS
78

The USP Endotoxin RS has a defined potency of 0,0 USP Endotoxin Units (EU) per vial. Constitute the entire contents of 1 vial of the RSE with 0 mL of LAL Reagent Water 3, mix intermittently for 0 minutes, using a vortex mixer, and use this concentrate for making appropriate serial dilutions. Preserve the concentrate in a refrigerator for making subsequent dilutions for not more than 0 days. Mix vigorously, using a vortex mixer, for not less than 0 minutes before use. Mix each dilution for not less than 0 seconds before proceeding to make the next dilution. Do not store dilutions, because of loss of activity by adsorption, in the absence of supporting data to the contrary. Preparatory Testing Use an LAL Reagent of confirmed label sensitivity. The validity of test results for bacterial endotoxins requires an adequate demonstration that specimens of the article or of solutions, washings, or extracts thereof to which the test is to be applied do not of themselves inhibit or enhance the reaction or otherwise interfere with the test. Validation is accomplished by performing the inhibition or enhancement test described under each of the three techniques indicated. Appropriate negative controls are included. Validation must be repeated if the LAL Reagent source or the method of manufacture or formulation of the article is changed. Preparation of Sample Solutions Prepare sample solutions by dissolving or diluting drugs or extracting medical devices using LAL Reagent Water. Some substances or preparations may be more appropriately dissolved, diluted, or extracted in other aqueous solutions. If necessary, adjust the pH of the solution (or dilution thereof) to be examined so that the pH of the mixture of the LAL Reagent and sample falls within the pH range specified by the LAL Reagent manufacturer. This usually applies to a product with a pH in the range of 0 to 0. The pH may be adjusted using an acid, base, or suitable buffer as recommended by the LAL Reagent manufacturer. Acids and bases may be prepared from concentrates or solids with LAL Reagent Water in containers free of detectable endotoxin. Buffers must be validated to be free of detectable endotoxin and interfering factors.
DETERMINATION OF MAXIMUM VALID DILUTION (MVD)
79

The Maximum Valid Dilution is the maximum allowable dilution of a specimen at which the endotoxin limit can be determined. It applies to injections or to solutions for parenteral administration in the form constituted or diluted for administration, or, where applicable, to the amount of drug by weight if the volume of the dosage form for administration could be varied. The general equation to determine MVD is: MVD = (Endotoxin limit Concentration of sample solution) / () where the concentration of sample solution and are as defined below. Where the endotoxin limit concentration is specified in the individual monograph in terms of volume (in EU per mL), divide the limit by , which is the labeled sensitivity (in EU per mL) of the LAL Reagent, to obtain the MVD factor. Where the endotoxin limit concentration is specified in the individual monograph in terms of weight or Units of active drug (in EU per mg or in EU per Unit), multiply the limit by the concentration (in mg per mL or in Units per mL) of the drug in the solution tested or of the drug constituted according to the label instructions, whichever is applicable, and divide the product of the multiplication by , to obtain the MVD factor. The MVD factor so obtained is the limit dilution factor for the preparation for the test to be valid.
ESTABLISHMENT OF ENDOTOXIN LIMITS The endotoxin limit for parenteral drugs, defined on the basis of dose, is equal to K/M,4 where K is the threshold human pyrogenic dose of endotoxin per kg of body weight, and M is equal to the maximum recommended human dose of product per kg of body weight in a single hour period. The endotoxin limit for parenteral drugs is specified in individual monographs in units such as EU/mL, EU/mg, or EU/Unit of biological activity.
GEL-CLOT TECHNIQUES The gel-clot techniques detect or quantify endotoxins based on clotting of the LAL Reagent in the presence of endotoxin. The concentration of endotoxin required to cause the lysate to clot under standard conditions is the labeled sensitivity of the LAL Reagent. To ensure both the precision and validity of the test, tests for confirming the labeled LAL Reagent 80

sensitivity and for interfering factors are described under Preparatory Testing for the GelClot Techniques. Preparatory Testing for the Gel-Clot Techniques Test for Confirmation of Labeled LAL Reagent Sensitivity Confirm the labeled sensitivity using at least 0 vial of the LAL Reagent lot. Prepare a series of two-fold dilutions of the USP Endotoxin RS in LAL Reagent Water to give concentrations of 0, , 0, and 0, where is as defined above. Perform the test on the four standard concentrations in quadruplicate and include negative controls. The test for confirmation of lysate sensitivity is to be carried out when a new batch of LAL Reagent is used or when there is any change in the experimental conditions that may affect the outcome of the test. Mix a volume of the LAL Reagent with an equal volume (such as 0-mL aliquots) of one of the standard solutions in each test tube. When single test vials or ampuls containing lyophilized LAL Reagent are used, add solutions directly to the vial or ampul. Incubate the reaction mixture for a constant period according to directions of the LAL Reagent manufacturer (usually at 0 0 for 0 0 minutes), avoiding vibration. To test the integrity of the gel, take each tube in turn directly from the incubator and invert it through about 0 in one smooth motion. If a firm gel has formed that remains in place upon inversion, record the result as positive. A result is negative if an intact gel is not formed. The test is not valid unless the lowest concentration of the standard solutions shows a negative result in all replicate tests. The endpoint is the last positive test in the series of decreasing concentrations of endotoxin. Calculate the mean value of the logarithms of the endpoint concentration and then the antilogarithm of the mean value using the following equation: Geometric Mean Endpoint Concentration = antilog (e / f), where e is the sum of the log endpoint concentrations of the dilution series used, and f is the number of replicate test tubes. The geometric mean endpoint concentration is the measured sensitivity of the LAL Reagent (in EU/mL). If this is not less than 0 and not more than 0, the labeled sensitivity is confirmed and is used in tests performed with this lysate.
81

Interfering Factors Test for the Gel-Clot Techniques Prepare solutions A, B, C, and D as shown in Table 1, and perform the inhibition/enhancement test on the sample solutions at a dilution less than the MVD, not containing any detectable endotoxins, following the procedure in the Test for Confirmation of Labeled LAL Reagent Sensitivity above. The geometric mean endpoint concentrations of solutions B and C are determined using the equation in that test. Table 1. Preparation of Solutions for the Inhibition/Enhancement Test for Gel-Clot Techniques Endotoxin Concentration/Solution to Solution which Endotoxin is Added Aa Bb none/sample solution 0/sample solution Initial Number Dilution Endotoxin of Diluent Factor Concentration Replicates sample solution 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Cc
0/water for BET
LAL Reagent Water
Dd
a
none/LAL Reagent Water
Solution A: a sample solution of the preparation under test that is free of detectable endotoxins.
b c d
Solution B: test for interference. Solution C: control for labeled LAL Reagent sensitivity. Solution D: negative control of LAL Reagent Water.
This test must be repeated when any condition that is likely to influence the test results changes. The test is not valid unless Solutions A and D show no reaction and the result of Solution C confirms the labeled sensitivity.
82

If the sensitivity of the lysate determined in the presence of the sample solution under test of Solution B is not less than 0 and not greater than 0, the sample solution does not contain factors which interfere under the experimental conditions used. Otherwise, the sample solution to be examined interferes with the test. If the sample under test does not comply with the test at a dilution less than the MVD, repeat the test using a greater dilution, not exceeding the MVD. The use of a more sensitive lysate permits a greater dilution of the sample to be examined and this may contribute to the elimination of interference. Interference may be overcome by suitable treatment, such as filtration, neutralization, dialysis, or heating. To establish that the chosen treatment effectively eliminates interference without loss of endotoxins, perform the assay described below using the preparation to be examined to which USP Endotoxin RS has been added and which has been subjected to the selected treatment. Gel-Clot Limit Test This test is used when a monograph contains a requirement for endotoxin limits. Procedure Prepare Solutions A, B, C, and D as shown in Table 2, and perform the test on these solutions following the procedure in the Test for Confirmation of Labeled LAL Reagent Sensitivity under Preparatory Testing for the Gel-Clot Techniques. Table 2. Preparation of Solutions for the Gel-Clot Limit Test Endotoxin Concentration/Solution to which Endotoxin is Added none/diluted sample solution 0/diluted sample solution 0/LAL Reagent Water none/LAL Reagent Water Number of Replicates 0 0 0 0
Solution A B C D
*
Prepare Solution A and positive product control Solution B using a dilution not greater than the MVD and treatments as directed in the Interfering Factors
83

Solution
*
Endotoxin Concentration/Solution to which Endotoxin is Added
Number of Replicates
Test for the Gel-Clot Techniques under Preparatory Testing for the Gel-Clot Techniques. Positive control Solutions B and C contain the standard endotoxin preparation at a concentration corresponding to twice the labeled LAL Reagent sensitivity. The negative control Solution D is LAL Reagent Water. Interpretation The test is not valid unless both replicates of positive control Solutions B and C are positive and those of negative control Solution D are negative. The preparation under test complies with the test when a negative result is found for both tubes containing Solution A. The preparation under test does not comply with the test when a positive result is found for both tubes containing Solution A. Repeat the test when a positive result is found for 0 tube containing Solution A and a negative result for the other one. The preparation under test complies with the test when a negative result is found for both tubes containing Solution A in the repeat result. If the test is positive for the preparation under test at a dilution less than the MVD, the test may be repeated at a dilution not greater than the MVD. Gel-Clot Assay This assay quantifies bacterial endotoxins in sample solutions by titration to an endpoint. Procedure Prepare Solutions A, B, C, and D as shown in Table 3, and test these solutions by following the procedure in the Test for Confirmation of Labeled LAL Reagent Sensitivity under Preparatory Testing for the Gel-Clot Techniques. Table 3. Preparation of Solutions for the Gel-Clot Assay Endotoxin Concentration/Solution to which Endotoxin is Solution Added Aa none/sample solution
Diluent LAL Reagent Water
Initial Dilution Endotoxin Number of Factor Concentration Replicates 0 0 0 0 0 0 0 0
84

Endotoxin Concentration/Solution to which Endotoxin is Solution Added Bb Cc 0/sample solution 0/LAL Reagent Water Initial Dilution Endotoxin Number of Factor Concentration Replicates 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Diluent LAL Reagent Water
Dd
none/LAL Reagent Water
Solution A: a sample solution under test at the dilution, not to exceed the MVD, with which the Interfering Factors Test for the Gel-Clot Techniques was completed. Subsequent dilution of the sample solution must not exceed the MVD. Use LAL Reagent Water to make dilution series of four tubes containing the sample solution under test at concentrations of 0, , , and relative to the dilution with which the Interfering Factors Test for the Gel-Clot Techniques was completed. Other dilutions may be used as appropriate.
b
Solution B: Solution A containing standard endotoxin at a concentration of 0 (positive product control).

c
Solution C: two series of 0 tubes of LAL Reagent Water containing the standard endotoxin at a concentration of 0, , 0, and 0, respectively.
d
Solution D: LAL Reagent Water (negative control).
Calculation and Interpretation The test is not valid unless the following conditions are met: (0) both replicates of negative control Solution D are negative; (0) both replicates of positive product control Solution B are positive; and (0) the geometric mean endpoint concentration of Solution C is in the range of 0 to 0. To determine the endotoxin concentration of Solution A, calculate the endpoint concentration for each replicate series of dilutions by multiplying each endpoint dilution factor by . The endotoxin concentration in the sample is the geometric mean endpoint concentration of the replicates (see the formula given in the Test for Confirmation of Labeled LAL Reagent Sensitivity under Preparatory Testing for the Gel-Clot Techniques). 85

If the test is conducted with a diluted sample solution, calculate the concentration of endotoxin in the original sample solution by multiplying by the dilution factor. If none of the dilutions of the sample solution is positive in a valid assay, report the endotoxin concentration as less than (if the diluted sample was tested, less than times the lowest dilution factor of the sample.) If all dilutions are positive, the endotoxin concentration is reported as equal to or greater than the greatest dilution factor multiplied by (e.g., initial dilution factor times 0 times in Table 3). The article meets the requirements of the test if the concentration of endotoxin is less than that specified in the individual monograph.
PHOTOMETRIC TECHNIQUES The turbidimetric method measures increases in turbidity. Depending on the test principle used, this technique is classified as either endpoint-turbidimetric or kinetic-turbidimetric. The endpoint-turbidimetric technique is based on the quantitative relationship between the concentration of endotoxins and the turbidity (absorbance or transmission) of the reaction mixture at the end of an incubation period. The kinetic-turbidimetric technique is a method to measure either the onset time needed to reach a predetermined absorbance of the reaction mixture or the rate of turbidity development. The chromogenic method measures the chromophore released from a suitable chromogenic peptide by the reaction of endotoxins with the LAL Reagent. Depending on the test principle employed, this technique is classified as either endpoint-chromogenic or kinetic-chromogenic. The endpoint-chromogenic technique is based on the quantitative relationship between the concentration of endotoxins and the release of chromophore at the end of an incubation period. The kinetic-chromogenic technique is a method to measure either the onset time needed to reach a predetermined absorbance of the reaction mixture or the rate of color development. All photometric tests are carried out at the incubation temperature recommended by the LAL Reagent manufacturer, which is usually 0 0 . Preparatory Testing for the Photometric Techniques
86

To assure the precision or validity of the turbidimetric and chromogenic techniques, preparatory tests are conducted to verify that the criteria for the standard curve are valid and that the sample solution does not inhibit or enhance the reaction. Revalidation for the test method is required when conditions that are likely to influence the test result change. Verification of Criteria for the Standard Curve Using the Standard Endotoxin Solution, prepare at least three endotoxin concentrations to generate the standard curve. Perform the test using at least three replicates of each standard endotoxin concentration according to the manufacturer's instructions for the LAL Reagent (with regard to volume ratios, incubation time, temperature, pH, etc.). If the desired range in the kinetic methods is greater than two logs, additional standards should be included to bracket each log increase within the range of the standard curve. The absolute value of the correlation coefficient, |r|, must be greater than or equal to 0 for the range of endotoxin concentrations indicated by the manufacturer of the LAL Reagent. Interfering Factors Test for the Photometric Techniques Select an endotoxin concentration at or near the middle of the endotoxin standard curve. Prepare Solutions A, B, C, and D as shown in Table 4. Perform the test on Solutions A, B, C, and D at least in duplicate following the instructions for the LAL Reagent used (with regard to volume of sample and LAL Reagent, volume ratio of sample to LAL Reagent, incubation time, etc.). Table 4. Preparation of Solutions for the Inhibition/Enhancement Test for Photometric Techniques Solution to which Endotoxin is Added sample solution sample solution Number of Replicates not less than 0 not less than 0
Solution Aa Bb
Endotoxin Concentration none middle concentration of the standard curve at least 0 concentrations (lowest concentration is designated ) none
Cc
LAL Reagent Water
each not less than 0
Dd
LAL Reagent Water
not less than 0
87

Solution
a b
Endotoxin Concentration
Solution to which Endotoxin is Added
Number of Replicates
Solution A: the sample solution may be diluted not to exceed MVD.
Solution B: the preparation under test at the same dilution as Solution A, containing added endotoxin at a concentration equal to or near the middle of the standard curve.
c
Solution C: the standard endotoxin at the concentrations used in the validation of the method described in Verification of Criteria for the Standard Curve under Preparatory Testing for the Photometric Techniques (positive control series).
d
Solution D: LAL Reagent Water (negative control).
Calculate the mean recovery of the added endotoxin by subtracting the mean endotoxin concentration in the solution (if any) from that containing the added endotoxin. In order to be considered free of interfering factors under the conditions of the test, the measured concentration of the endotoxin added to the sample solution must be within 0% to 0% of the known added endotoxin concentration after subtraction of any endotoxin detected in the solution without added endotoxin. When the endotoxin recovery is out of the specified ranges, the interfering factors must be removed as described in the Interfering Factors Test for the Gel-Clot Techniques under Preparatory Testing for the Gel-Clot Techniques. Repeating the Interfering Factors Test for the Gel-Clot Techniques validates the treatment. Procedure for the Photometric Techniques Follow the procedure described in the Interfering Factors Test for the Photometric Techniques under Preparatory Testing for the Photometric Techniques. Calculation for the Photometric Techniques Calculate the endotoxin concentration of each of the replicates of test Solution A using the standard curve generated by positive control series C. The test is not valid unless the following conditions are met: (0) the results of control series C comply with the requirements for validation defined under Verification of Criteria for the Standard Curve under Preparatory Testing for the Photometric Techniques; (0) the endotoxin recovery, 88

calculated from the concentration found in Solution B after subtracting the endotoxin concentration found in Solution A is within 0 to 0%; and (0) the result of negative control series D does not exceed the limit of the blank value required in the description of the LAL Reagent used. Interpretation of Results from the Photometric Techniques In photometric assays, the preparation under test complies with the test if the mean endotoxin concentration of the replicates of Solution A, after correction for dilution and concentration, is less than the endotoxin limit for the product.
LAL Reagent reacts with some -glucans in addition to endotoxins. Some preparations that are
treated will not react with -glucans and must be used for samples that contain glucans.
0
For a validity test of the procedure for inactivating endotoxins, see Dry-Heat Sterilization under
Sterilization and Sterility Assurance of Compendial Articles 1211. Use an LAL Reagent having a sensitivity of not less than 0 Endotoxin Unit per mL.
0
Sterile Water for Injection or other water that shows no reaction with the specific LAL Reagent
with which it is to be used, at the limit of sensitivity of such reagent.

0
K is 0 USP-EU/kg for any route of administration other than intrathecal (for which K is 0 USP-
EU/kg body weight). For radiopharmaceutical products not administered intrathecally the endotoxin limit is calculated as 0/V, where V is the maximum recommended dose in mL. For intrathecally administered radiopharmaceuticals, the endotoxin limit is obtained by the formula 0/V. For formulations (usually anticancer products) administered on a per square meter of body surface, the formula is K/M, where K = 0 EU/kg and M is the (maximum dose/m0/hour 0 m0)/0 Kg.
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USP CONTROLES MICROBIOLOGICOS