Professional Documents
Culture Documents
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.
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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
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. ]
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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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
00
0 to 0 hours
0 to 0 days
00
0 to 0 hours
0 to 0 days
00
0 to 0 hours
0 to 0 days
00
0 to 0 days 0 to 0 days
Organism
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:
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Available from American Type Culture Collection, 0 University Boulevard, Manassas, VA 0-0
(http://www.atcc.org).
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
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
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:
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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
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
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
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
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
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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
0g 0g 0g
Sodium Chloride
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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
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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
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
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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
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
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0g 0 mL
0g 0g 0g 0 mg 0 mL
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
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
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 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
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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
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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.
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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
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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
Gram Stain Positive cocci (in clusters) Positive cocci (in clusters) Positive cocci (in clusters)
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
Yellowish
Positive
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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.
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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.*
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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
34
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
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
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
Papaic Digest of Soybean Meal 0 g Sodium Chloride Dibasic Potassium Phosphate Dextrose (C0H0O0H0O) 37 0g 0g 0/0 g
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
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
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
ATCC 0, CIP 0, NCTC 0, NCIMB 0 ATCC 0, CIP 0, NCIMB 0 ATCC 0, NCIMB 0, CIP 0
An alternative to Staphylococcus aureus is Bacillus subtilis (ATCC 0). An alternative microorganism is Micrococcus luteus (Kocuria rhizophila),
ATCC 0.
0
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.]
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
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
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)
42
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)
43
Quantity per Container Sutures and other individually packaged single-use material Other medical devices
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
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
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
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
and filter. The diluent may contain suitable neutralizing substances and/or appropriate inactivating substances, for example, in the case of antibiotics.
46
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
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
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
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
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
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
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
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
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
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
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
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
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
0 mL
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
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
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
pH after sterilization: 0 0.
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
pH after sterilization: 0 0.
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
58
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
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
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
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
0 mg
B. 0
0 g
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)
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
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
0 mg
B. 0
0 g
Water
0 mg
Water
0 g
0 mg
Water
0 g
Erythromycin (CP)
0 mg
B. 0
0 g
63
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
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
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 mg
Water
0 g
Thiostrepton (T)
0U
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)
0 mg
Water
0 g
Tylosin (T)
0 mg
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
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
Number 0 0 0
Escherichia coli
0 0
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
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
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
0 to 0 0 hr.
0 to 0 0 hr.
0 to 0 0 to 0 hr.
Capreomycin
70
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.
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 0
0 0 0 0
0 0 0 0 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
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
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
75
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
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. ]
78
79
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
81
Cc
Dd
a
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
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
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
84
Dd
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 C: two series of 0 tubes of LAL Reagent Water containing the standard endotoxin at a concentration of 0, , 0, and 0, respectively.
d
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
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
Solution Aa Bb
Endotoxin Concentration none middle concentration of the standard curve at least 0 concentrations (lowest concentration is designated ) none
Cc
Dd
87
Endotoxin Concentration
Number of Replicates
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
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
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
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.
89
90