Methods of Analysis: 3. Biological methods: 3.

2 Test for sterility

This test for sterility is applicable only to parenteral preparations. For ophthalmic and other non-injectable preparations, surgical dressings, catgut and other surgical sutures, appropriate alternative test conditions must be chosen. Sterility testing should be carried out under strict conditions specifically designed to avoid microbial contamination of the material being tested, for example by using a laminar flow technique. Precautions should also be taken to ensure that any microorganisms expected to be revealed by the test remain unmodified. Adequate performance of the testing technique should be ensured periodically. A batch (i.e. one sterilized load, or a quantity of containers filled under aseptic conditions in one working session at one work station) is defined as a collection of sealed containers whose contents and physical aspect are homogeneous in every respect. The risk of microbial contamination is thus assumed to be equal for each container in the batch. Therefore, once a sample has been tested and is found to be free of contamination, the same degree of purity should apply to the whole batch. Assurance that this is the case depends strongly on compliance with Good Manufacturing Practices (GMP)1 throughout the entire manufacturing process.
1

WHO good manufacturing practices: main principals for pharmaceutical products. Quality assurance of pharmaceuticals. A compendium of guidelines and related materials, Volume 2, updated edition. Geneva, World Health Organization, 2004. However, many national authorities have incorporated good manufacturing practices of their own into their legislation, and these should be followed when available. Sampling Number of containers Minimum number of samples in the batch to be tested not more than 100 between 100 and 500 more than 500 Culture media Culture media that have proven to be the most suitable for use in sterility tests are: fluid sodium mercaptoacetate and soya-bean casein digest media. Fluid sodium mercaptoacetate (sodium thioglycolate) medium (culture medium Cm4) is primarily intended for the culture of anaerobic bacteria but will also detect aerobic bacteria. Soya-bean casein digest medium (culture medium Cm5) is primarily intended for the culture of aerobic bacteria but is also suitable for fungi. Other media may be used provided that they have been shown to sustain the 10% or 4 containers (whichever is greater) 10 containers 2% or 20 containers (whichever is less)

growth of a wide range of microorganisms and that they comply with the test for "effectiveness of the medium" in the presence of the preparation to be tested. The growth-promoting quality of each new batch of media should be tested as follows: take separate representative samples of the medium and add inocula of reference strains of appropriate microorganisms (e.g. Staphylococcus aureus ATCC 6538 P (NCIMB 8625, CIP 53.156), Bacillus subtilis ATCC 6633, Clostridium sporogenes ATCC 19404, Candida albicans ATCC 2091), each containing approximately 100 viable microorganisms. The media thus inoculated with the reference strains should then be incubated according to the conditions specified in the recommended procedure. The test media are satisfactory if clear evidence of growth appears in all inoculated media within 7 days. Antibacterial effects of the sample Before testing, possible antibacterial action of the substance or material under examination should be determined and the growth-promoting properties of the media in the presence and in the absence of the test sample should be compared. This comparison should be carried out under test conditions using reference strains, as described above in the section "Culture media". If the growth of the reference strains is delayed or weakened in the presence of the product, the antibacterial action of the product should be suppressed by means of filtration, dilution, or neutralization. The effectiveness of this process should be confirmed by repeating the test. Recommended procedures The test for sterility may be carried out by membrane filtration or by direct inoculation. The technique of membrane filtration is to be preferred whenever the nature of the product permits, i.e. for aqueous preparations able to be filtered, especially in large volumes, for alcoholic or oily preparations, and for preparations miscible with or soluble in aqueous or oily solvents that have not shown any antimicrobial effect when tested. Whichever test is used, the media should be observed at regular intervals throughout the specified incubation period. Membrane filtration Use membrane filters of assured quality, preferably certified by the manufacturer, with a nominal pore size not greater than 0.45 m. For example, use cellulose nitrate filters for aqueous, oily, and weakly alcoholic liquids and acetate filters for strongly alcoholic liquids. Use filter discs with a diameter of about 50 mm in a previously sterilized filter assembly. If filters of a different diameter are used, adjust the volumes of the dilutions and the washings accordingly. Before the test, filter a small quantity of a suitable sterile diluent, such as a 1 g/litre neutral solution of meat or casein peptone (culture media Cm1 or Cm5), through the prepared filtration apparatus. Transfer the contents of the container(s) to be examined to the apparatus. If possible, transfer the entire contents of the containers or the minimum quantity laid down for the test by direct inoculation. If necessary, dilute the product to be tested with the chosen sterile diluent. Carry out the filtration without delay. If the product to be tested has known antimicrobial properties, wash the membrane by filtering through it not less than three successive quantities of the chosen sterile diluent, each

of approximately 100 ml, with the addition of a suitable neutralizing substance. Oily liquids may be diluted before filtration with a suitable sterile solvent, such as isopropyl myristate, shown to have minimal antimicrobial effects. Preferably, transfer one membrane to each of the media used or, where this is not possible, transfer the one membrane cut aseptically into two equal parts to the different media. Incubate the culture media with the membranes for not less than 7 days at 30-35 C if it is intended mainly to detect bacteria and at 20-25 C if it is specifically intended to detect fungi. If the product or the treatment to which it has been subjected is suspect, a longer incubation time than 7 days may be necessary. Direct inoculation Transfer directly a suitable quantity (see the following tables) of the product under examination, taken from a sealed container, to culture media intended for the detection of aerobic and anaerobic bacteria as well as media for the detection of fungi. Liquids Quantity in the container less than 1 ml between 1 ml and 4 ml between 4 ml and 20 ml Quantity of sample needed entire contents of container half contents of container 2 ml

20 ml or more (including largevolume parenterals) 10% of contents Solids Quantity in the container Quantity of sample needed less than 50 mg entire contents of container

between 50 mg and 200 mg 50% of contents of container 200 mg or more 100 mg

There should be a sufficient quantity of culture medium to ensure that its nutritive properties are unaffected by the addition of the product under examination. In order to ensure homogeneous distribution and to eliminate antibacterial activity, unless otherwise indicated in the monograph, transfer the product under examination in such a way that liquids are diluted approximately 10-fold and solids approximately 100-fold.

In the case of an oily liquid, an emulsifying agent may be added to the culture medium, such as 0.5-1% m/v of polysorbate 80 or 0.1% m/v of (p-tert-octylphenoxy)polyoxyethanol. Other emulsifying agents shown not to have any antimicrobial action under test conditions may also be used in appropriate concentrations. Incubate the inoculated media for not less than 14 days. Maintain the temperature at 30-35 C for media mainly intended to detect bacteria and at 20-25 C for media specifically intended to detect fungi. Gently shake at regular intervals if the medium contains an oily liquid. If one medium is used to detect both aerobic and anaerobic bacteria, keep the shaking to a minimum in order to maintain anaerobic conditions. Interpretation of results If no microbial growth appears in any of the culture media by the end of the incubation period, the product complies with the test for sterility. If microbial growth appears in the initial test cultures and/or in subcultures, the organisms should be isolated and identified. A second test should then be made to verify the results. If there is evidence - from the material used, the kind of organisms detected, the monitoring or the control tests - that the test was carried out under inadequately aseptic conditions, the test is invalid and should be repeated with the same corresponding number of items and quantities. If the microbial growth is not due to lack of aseptic conditions, the test should be repeated on twice the number of containers used for the original test. If no microbial growth is detected, the product examined complies with the test for sterility. If microbial growth is detected in the second test, the product examined does not comply with the test for sterility.

Methods of Analysis: 3. Biological methods: 3.2 Test for sterility: 3.2.1 Test for sterility of non-injectable preparations
The methods are described under 3.2 Test for sterility. Certain dosage forms such as ophthalmic drops, ophthalmic ointments, topical semi-solid dosage forms to be applied to damaged skin, etc., require specific sampling plans and preparation of samples. For the membrane filtration method and for direct inoculation of the medium, determine the number of containers according to the sampling method proposed. The quantity taken should be not less than the minimum and not more than the maximum quantity given below. The quantity used for each medium, which should be taken from the mixed sample, is also specified in the table. Sampling plan Type of preparation Total pooled for each Quantity to be medium used

Membrane filtration method Aqueous solution 10 - 100ml 5 - 10ml equivalent to 0.5 - 1g

Preparations soluble in water, isopropyl myristate, or 1 - 10g other solvents

Direct inoculation method Liquid preparations Soluble preparations Insoluble preparations to be suspended or emulsified, e.g. creams and ointments 10 - 100ml 1 - 10g 1 - 10g 5 - 10ml equivalent to 0.5 - 1g equivalent to 0.5 - 1g

If the membrane filtration method is used for ointments and creams, the preparation being tested may require additional heating up to 40 C (or exceptionally up to 45 C with continuous stirring). The filtration process should be carried out as quickly as possible. If heat is applied and/or a solvent combination is used in the sample preparation, the method should first be validated to ensure that the specific expected bioburden is not affected by these conditions.

Methods of Analysis: 3. Biological methods: 3.2 Test for sterility: 3.2.2 Sterility testing of antibiotics
The test is designed to reveal the presence of contamination with live microorganisms in antibiotics intended for parenteral administration or for other sterile applications. Test conditions The test should be carried out under aseptic conditions in an area as free from contamination as it is possible to achieve by the use of disinfecting agents, germicidal lamps, and air filters. Germicidal lamps and disinfecting aerosols should not be used during actual testing operations. The test manipulations should be carried out in a filtered air environment or under a laminar flow hood, with operators dressed in sterilized, static-free clothing, including head and foot wear. The air pressure in the testing room should be greater than that of the exterior area. The performance of the laminar flow hood should be monitored by particulate count, settle plates, or slit-sampling devices, and the performance of the filters and germicidal lamps checked routinely. Membrane filtration apparatus

A suitable unit consists of a closed reservoir and a receptacle, separated by a properly supported membrane of appropriate porosity. Membranes generally suitable for sterility testing have a nominal porosity of 0.45 m, a diameter of approximately 47 mm, and a flow rate of 55-75 ml of water per minute at a pressure of 90 kPa (700 mm Hg). The entire unit should preferably be assembled and sterilized with the membrane in place, prior to use. If each entire membrane is to be cultured, at least 2 filter units should be set up. All air entering the filtering unit should be passed through an air-filter capable of removing microorganisms. Sampling Take the sample in such a manner as to be representative of the material to be tested. The amount taken should be sufficient to perform the tests and any repeat tests that may be required. The sampling should be carried out in such a way as to maintain intact the sterility of the material. Culture media The culture media used for sterility tests for bacteria and fungi should be capable of supporting the growth of a wide variety of microorganisms, with both aerobic and anaerobic growth characteristics, including the types found in the environment of the manufacturing operations. More than one culture medium will generally be needed to fulfil these criteria. The media that usually give satisfactory results are fluid mercaptoacetate (thioglycolate) medium (culture medium Cm4) and soybean-casein digest medium (culture medium Cm5). Any other media that are used, however, should have at least demonstrably equivalent growth-supporting properties. For testing the growth-supporting properties of each culture medium, strains of microorganisms should be used with exacting nutritive and aerobic-anaerobic requirements, in an inoculum containing only a small number of organisms (less than 100). The media should be incubated at the temperatures at which they will be used in the sterility test and growth should be evident after 24 hours. Each lot of dehydrated medium obtained from a specialized manufacturer or each lot of the medium prepared entirely in the laboratory should be tested for its growth-supporting properties, since not every lot may support the growth of microorganisms to the desired extent. Differences may be caused by the occasional presence of unsatisfactory components in a particular lot, or by the destruction of certain components by overheating or oversterilization of the medium. Recommended procedures Membrane filtration test procedure Aseptically transfer a suitable amount of the solid test material (0.3-6 g depending on the size of the container) into a sterile flask containing about 200 ml of peptone (1 g/l) TS1, stopper the flask and swirl to effect rapid dissolution. If the test material dissolves slowly or if the resulting solution will not filter rapidly, the volume of the solvent may be increased to not more than 400 ml. Immediately after the test material has dissolved, aseptically filter the

solution, with the aid of reduced pressure, through the membrane filter previously moistened with sterile water or with peptone (1 g/l) TS1. To speed up the process of filtration, the solution may be filtered using two filtering units simultaneously. To remove the residual antibiotic from the membrane, wash it with sufficient peptone (1 g/l) TS1 to which, if so indicated in the monograph (in the case of penicillin and cephalosporin antibiotics), sufficient penicillinase TS has been added. Upon completion of the filtration, divide the membrane aseptically into two approximately equal parts. Transfer one part of the membrane into a culture vessel (a test-tube is suitable) containing 50-100 ml of culture medium Cm4 (fluid mercaptoacetate (thioglycolate) medium) and the other part of the membrane into another culture vessel containing 50-100 ml of culture medium Cm5 (soybean-casein digest medium). A control test should be carried out at appropriate intervals to demonstrate that residual antibiotic activity is being reduced by the above described washing procedure to below the level that allows growth of an inoculum containing 50-100 microorganisms susceptible to the antibiotic tested. Direct test procedure Depending on the size of the container take from 1-10 portions, each 0.3 g of the test material, and aseptically transfer them into individual sterile vessels (test-tubes are suitable) containing 50-100 ml of culture medium Cm6 (fluid mercaptoacetate (thioglycolate) medium with penicillinase). Transfer portions of similar size to another set of individual sterile vessels containing 50-100 ml of culture medium Cm7 (soybean-casein digest medium with penicillinase). Check the ability of the penicillinase contained in the medium to inactivate all the penicillin in the test material by adding to one vessel containing culture medium Cm6 the amount of material taken from one container under test. Next add 1.0 ml of a dilution containing 50-100 microorganisms of a suitable strain (Staphylococcus aureus, ATCC 6538-P is suitable) in culture medium Cm4. Typical microbial growth must be observable after 24 hours of incubation at 30-32 C. Incubation Incubate for 7 days the vessels containing fluid mercaptoacetate (thioglycolate) media (culture media Cm4 and Cm6) at 30-32 C and the vessels containing soybean-casein digest media (media Cm5 and Cm7) at 22-25 C. In the direct test procedure, gently agitate the vessels at least once a day or until complete dissolution occurs. If other culture media are used, the incubation temperature and the period of incubation may have to be appropriately modified. Examine inoculated culture vessels at regular intervals and on the last day of incubation for evidence of microbial growth. If such growth is observed it should be confirmed by microscopic examination. It is desirable that the incubation apparatus be equipped with a continuous temperature-recording device. Interpretation of test results

If no evidence of growth is found in any of the culture vessels, except in the positive growth control, the material meets the requirements of the test. If, however, evidence of growth is found, a repeat test may be performed. If no evidence of growth is then found in any of the culture vessels, except in the positive growth control, the material meets the requirement of the test. The material fails to pass the test if growth occurs in the repeat test. The distinction between failure of the product to pass the test and a possible invalidity of the test procedure requires the competent judgement of an expert.

Pharmaceutical Sterility Testing

Essential things to know
By Steven Richter

Sterility testing of pharmaceutical articles is required during the sterilization validation process as well as for routine release testing. USP1 requirements employ sterility testing as an official test to determine suitability of a lot. An understanding of sterility testing is beneficial in terms of designing a validation process. The need to provide adequate and reliable sterility test data is an important quality assurance issue. Sterility testing is a very tedious and artful process that must be performed by trained and qualified laboratory personnel. The investigation of sterility test failures is a process that requires attention to environmental data as well as many other factors including training and sample difficulty. This paper presents the general concepts and problems associated with sterility testing as well as the various testing methodologies. Most USP <71> sections are harmonized with the EP/JP. Sterility testing is an essential part of every sterilization validation. Sterility testing is an extremely difficult process that must be designed and executed so as to eliminate false positive results. False positive results are generally due to laboratory contamination from the testing environment or technician error. The testing environment must be designed to meet the requirements of the United States Pharmacopeia (USP) in terms of viable microbial air and surface counts. Growth media used in sterility testing must be meticulously prepared and

tested to ensure its ability to support microbial growth. Procedures for sampling, testing, and follow-up must be defined in the validation procedures.

Sampling Plans
The official test, the USP (Volume 30) recommends testing 40 units per production lot. A reprint of Table 2 "Minimum Quantity to be Used for Each Medium2" is on the next page. Some of the quantities are not harmonized with the EP/JP volumes.3

For combination products, the ISO 11137/111354 standards recommend various sterilization validation sampling plans based on lot size and validation method. In cases where small lots (>1000) are manufactured, the sampling size depends on lot size.

Environmental Concerns Related to Sterility Testing

The sterility test environment is described in USP General Informational Chapter <1211>. The environment should be as stringently controlled as an aseptic processing environment. An aseptic processing environment (clean room) is used to dispense sterile pharmaceuticals into presterilized containers. A clean room is generally a room that delivers laminar flow air which has been filtered through microbial retentive High Efficiency Particulate Air (HEPA) filters. The room is maintained under positive pressure and has specifications for room air changes per hour. An environment used for sterility testing should be similar in design to an aseptic

processing environment; there should be an anteroom for gowning and a separate area for the actual sterility testing. The testing area should meet ISO Class 5 particulate control requirements (specified in USP chapter (1116)). Sterility testing should not be carried out under a laminar flow hood located within a room that is not maintained as ISO Class 5. Along with particulate testing in the environment, the laboratory must test for viable bacterial and fungal organisms ubiquitous to it. The sterility test technician must be suitably gowned in sterile garments that prevent microbial shedding into the room. The room should be validated in terms of particulate and microbial levels. The laboratory must have a validation and training program for gowning and sterility testing. Our validation programs require that technicians consecutively test 40 simulated samples for both membrane filtration and direct immersion methods without a false positive test result under less than ideal environmental conditions. Isolator technology is utilized to create a sterile environment for one to test pharmaceutical articles. The validation required to qualify an isolator is extensive. The isolators are generally sterilized using chemical sterilization. Many issues surround the robustness of the sterilization process. Qualifying and maintaining an isolator system for sterility testing may require extensive work. In testing pharmaceutical articles in a closed system such as SteritestTM, an isolator may not be the best cost approach to the environmental concerns. Most environmental concerns can be obviated by standard aseptic processing GMP's.5

Methodologies
The United States Pharmacopeia is a compilation of validated methods and official monographs for pharmaceuticals and medical devices. IT is broken down into the following sections: Monographs, General Informational Chapters, and General Requirements. General Informational Chapters <1000> series are not legal requirements. The Sterility Test (USP Section <71>) is categorized under General Requirements and is therefore a legal requirement. For combination products, the ISO radiation sterilization microbial methods (11737-2 1998)6 describes a sterility test which is a modification for the USP method. This test is specific for the detection of aerobic organisms that have been exposed to sub-lethal sterilization cycles. This ISO sterility test method is recommended for the validation of both gamma and electron beam sterilization processes. The method of choice for EO7 sterilized products is the official USP <71> procedure.

Processes
Prior to actual sterility testing, it is prudent to send an example sample to the testing laboratory so the laboratory can determine the appropriate testing procedure. Each product should have a unique procedural specification for testing. The procedure should be very specific in terms of which items (or vials/syringes) to test. The procedure must indicate the Sample Item Portion (SIP). The Sample Item Portion is the percentage of the complete product tested. Since medical devices come in all shapes and sizes, it is very difficult to test large and cumbersome medical devices in their entirety. Therefore, the test laboratory will

determine a Sample Item Portion which is a portion of the sample expressed in fractional terms (i.e. 0.1 for 10% of the sample). This number is used in gamma and electron beam dose setting methods. The SIP portion should be validated by sterility testing. Combination products have unique challenges. A combination product is defined as one that has a drug component with medical device. For example, a drug coated stent. The agency's Office of Combination Products (OCP) would determine which regulatory branch (CDRH, CDER or CBER) is officiating the product. Official USP sterility testing of combination products is required for all sterile drug products. The drug product component applied aseptically creates the largest challenge to laboratory personnel. Biologics must be aseptically processed and cannot be terminally sterilized. In the near future, we will see more biologics that are combination products. Combination products sterilized by radiation are generally handled as medical devices following the ISO 11137 standard. For the most part, pharmaceutical GMPs would take precedent over 820 QSR8 requirements with all combination products. The more robust GMP9 requirement would assure reduced bioburden counts and consistent microbial populations during manufacturing. The USP <71> Sterility Test contains two qualifying assays which must be performed prior to sterility testing. They are the "Suitability Test" (Growth Promotion Test) and the "Validation Test" (Bacteriostasis and Fungistasis Test). The Suitability Test is used to confirm that each lot of growth media used in the sterility test procedure will support the growth of fewer than 100 viable microorganisms. If the media cannot support the growth of the indicator organisms, then the test fails. Secondly, a portion of each media lot must be incubated and assessed for sterility according to the incubation parameters (time, temperature) established by the method. If the media is found to be nonsterile, then the test fails. The Validation Test is used to determine if the test sample will inhibit the growth of microorganisms in the test media. Stasis, in terms of microbiology, is defined as the inability of a microorganism to grow and proliferate in microbiological media. Media that is bacteriostatic does not necessarily kill bacteria; it simply may retard bacterial growth and proliferation. The Validation Test must be performed on each product prior to and/or during sterility testing. This test determines if the media volumes are valid for the particular product. Some medical products contain bacteriostatic and fungistatic compounds that may require special procedures and special media for testing. This test is similar to the Suitability Test described above, however, the product sample is placed in the media along with the microorganisms. Microbial growth in the presence of the test samples is compared to controls without test samples. If microbial growth is present in the sample and control containers, then the test is valid. The next step is to proceed to actual sterility testing. Suitability, validation and sterility tests can be performed simultaneously. The USP describes three general methods for sterility testing: 1) Membrane Filtration, 2) Direct Transfer (Product Immersion); and 3) Product Flush.

Membrane Filtration Sterility Testing

The Membrane Filtration Sterility Test is the method of choice for pharmaceutical products. It is not the method of choice for medical devices; the FDA may question the rationale behind using the membrane filtration test over the direct transfer test for devices. An appropriate use of this test is for devices that contain a preservative and are bacteriostatic and/or fungistatic under the direct transfer method. With membrane filtration, the concept is that the microorganisms will collect onto the surface of a 0.45 micron pore size filter. This filter is segmented and transferred to appropriate media. The test media are fluid thioglycollate medium (FTM) and soybean casein digest medium (SCDM). FTM is selected based upon its ability to support the growth of anaerobic and aerobic microorganisms. SCDM is selected based upon its ability to support a wide range of aerobic bacteria and fungi (i.e. yeasts and molds). The incubation time is 14 days. Since there are many manipulations required for membrane filtration medical device sterility testing, the propensity for laboratory contamination is high. Therefore, in an open system, more sterility failures are expected when using this method. A closed system is recommended for drugs and small devices or combination products. Most pharmaceutical articles are tested using a closed system. In closed systems, the propensity for extrinsic contamination is very low.

Direct Transfer Sterility Testing

Combination products: This method is the method of choice for medical devices because the device is in direct contact with test media throughout the incubation period. Viable microorganisms that may be in or on a product after faulty/inadequate sterilization have an ideal environment within which to grow and proliferate. This is especially true with damaged microorganisms where the damage is due to a sub-lethal sterilization process. All microorganisms have biological repair mechanisms that can take advantage of environmental conditions conducive to growth. The direct transfer method benefits these damaged microorganisms. The entire product should be immersed in test fluid. With large devices, patient contact areas should be immersed. Large catheters can be syringe filled with test media prior to immersion. Cutting catheter samples to allow for complete immersion is the method of choice. The USP authors understand that appropriate modifications are required due to the size and shape of the test samples. The method requires that the product be transferred to separate containers of both FTM and SCDM. The product is aseptically cut, or transferred whole, into the media containers. The test article should be completely immersed in the test media. The USP limits the media volume to 2500 ml. After transferring, the samples are incubated for 14 days.

Product Flush Sterility Testing

Combination products: The product flush sterility test is reserved for products that have hollow tubes such as transfusion and infusion assemblies where immersion is impractical and where the fluid pathway is labeled as sterile. This method is easy to perform and requires a modification of the FTM media for small lumen devices. The products are flushed with fluid D and the eluate is membrane filtered and placed into FTM and SCDM. This method is not generally used.

Bulk Drug Products / Biologics and Pharmaceuticals

Bulk Pharmaceuticals (APIs) are tested for sterility per USP 71 prior to release to the manufacturing processes. Bulk Biologics are tested according to 21 CFR 610.12 for sterility testing. This method requires one media (FTM). The sample test sizes are listed in the document. Volumes are no less than 10 ml.10

Interpretation of Sterility Test Results

The technician must be trained in the method of detecting growth during the incubation period. Growth is determined by viewing the media, which is generally clear and transparent, against a light source. Turbid (cloudy) areas in the media are indicative of microbial growth. Once growth is detected, the suspect vessel is tested to confirm that the turbidity present is due to microorganisms and not due to disintegration of the sample; sometimes samples produce turbidity because of particulate shedding or chemical reactions with the media. Once a suspect container has been tested, it should be returned to the incubator for the remainder of the incubation period. Samples that render the media turbid are transferred on Day 14 of the test and incubated for four days. Growth positive samples require further processing such as identification and storage.

Sterility Test Failure Investigation

For every positive sterility test (OOS), the laboratory should perform an OOS investigation to determine the validity of the positive growth. This investigation encompasses the following items: 1. 2. 3. 4. 5. 6. clean room environmental test (EER) data; media sterilization records; technician training records; the relative difficulty of the test procedure; control data (open and closed media controls); technician sampling data (microbial counts on gloves and/or garments post testing).

The USP allows for a re-test of the product if persuasive evidence exists to show that the cause of the initial sterility failure was induced by the laboratory. Identification and speciation of the isolate(s) is a significant contributing factor to the final decision. If the First Stage sterility test can be invalidated by the laboratory, then the USP allows for Second Stage sterility testing. Second Stage sterility testing requires double the original number of samples tested. The Second Stage test can be repeated if evidence exists invalidating the test due to a laboratory error as above. A detailed investigation may uncover circumstantial evidence to support a final decision. It is recommended that sterilization cycle data, environmental data, and bioburden data be

reviewed prior to making any decision to release product. It is recommended that medical device manufacturers qualify the test procedure with nonsterile samples. The probability of a false positive can be calculated using John Lee's formula.11 The formula is based upon sample container diameter, amount of time container is left open and the room particulate count. Sterility testing requires high levels of control with regards to GMPs, Good Laboratory Practices12, environment (aseptic clean room ISO class 5 or better), and employee practices. It is essential that meticulous technique be employed in the practice of sterility testing. Sterility testing is an integral part of sterilization validation as well as a routine quality control. Generally, false positive results are uncommon in testing drug products using a closed system. Combination products have challenges that should be planned into a robust QA program.

References
1. The United States Pharmacopeia, 30th Revision, The United States Pharmacopeial Convention: 2008 2. USP 30 Table 2 Minimum Quantity to be Used for Each Medium 3. USP 30 Table 3: Minimum Number of Articles to be Tested in Relation to the Number of Articles in the Batch 4. ISO 11137 Sterilization of health care products Radiation Part 2 2006: Establishing the sterilization dose 5. FDA Guidelines 2004 "Guidance for Industry Sterile Drug Products by Aseptic Processing, Current Good Manufacturing Practices," September, 2004 6. ISO 11737 ANSI/AAMI/ISO 11737-2 1998 Sterilization of Medical Devices Microbiological Methods Part 2, Tests of Sterility Performed in the Validation of a Sterilization Process 7. ISO 11135 1994 Medical Devices Validation and Routine Control of Ethylene Oxide Sterilization 8. Code of Federal Regulations Title 21/Chapter I/Part 820, "Quality Systems Requirements: General," 2006 9. GMPs CFR 201 Title 21 2006 10. 21 CFR Part 610.12 Bulk Biologics 11. Lee, John Y. "Investigation Sterility Test Failures" Pharmaceutical Technology, February 1990 12. Code of Federal Regulations Title 21/Chapter I/Part 58, "Good Laboratory Practice for Nonclinical Laboratory Studies," 2006

Steven Richter, Ph.D. is president and chief scientific officer of Microtest Laboratories, Inc. With his expertise in Sterilization Sciences, Dr. Richter founded Microtest in 1984 after a distinguished career at the FDA. For more information, contact: info@microtestlabs.com, (800) 631-1680

Search USP29 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 by an alternative procedure.

For additional information on sterility 1211 .

testing, see Sterilization and Sterility Assurance of Compendial Articles 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 Sodium Chloride Dextrose (C6H12O6H2O) 0.5 g 2.5 g 5.5/5.0 g

Agar, granulated (moisture content not exceeding 15%) 0.75 g Yeast Extract (water-soluble) Pancreatic Digest of Casein Sodium Thioglycollate or Thioglycolic Acid 5.0 g 15.0 g 0.5 g 0.3 mL

Resazurin Sodium Solution (1 in 1000), freshly prepared 1.0 mL Purified Water 1000 mL 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 1 N sodium hydroxide so that, after sterilization, the solution will have a pH of 7.1 0.2. 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 2 and 25 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 32.5 2.5 . 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 7.1 0.2. Incubate under anaerobic conditions for the duration of the incubation period. Alternative Fluid Thioglycollate Medium is to be incubated at 32.5 2.5 . SoybeanCasein Digest Medium Pancreatic Digest of Casein Sodium Chloride Dibasic Potassium Phosphate Dextrose (C6H12O6H2O) Purified Water 17.0 g 5.0 g 2.5 g 2.5/2.3 g 1000 mL

Papaic Digest of Soybean Meal 3.0 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 1 N sodium hydroxide so that, after sterilization, it will have a pH of 7.3 0.2. Filter, if necessary to clarify, dispense into suitable containers, and sterilize using a validated procedure. Store at a temperature between 2 and 25 in a sterile well-closed container, unless it is intended for immediate use. SoybeanCasein Digest Medium is to be incubated at 22.5 2.5 . 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. [NOTESupplemented 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, -lactamase media can

following either method under Validation Test, using less than 100 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. Test and the Validation Test Aerobic bacteria Staphylococcus aureus Bacillus subtilis Pseudomonas aeruginosa Anaerobic bacterium Clostridium sporogenes Fungi Candida albicans Aspergillus niger ATCC 10231, IP 48.72, NCPF 3179 ATCC 16404, IP 1431.83, IMI 149007
3 2 1

Table 1. Strains of the Test Microorganisms Suitable for Use in the Growth Promotion

ATCC 6538, CIP 4.83, NCTC 10788, NCIMB 9518 ATCC 6633, CIP 52.62, NCIMB 8054 ATCC 9027, NCIMB 8626, CIP 82.118

ATCC 19404, CIP 79.3, NCTC 532 or ATCC 11437

1 An alternative to Staphylococcus aureus is Bacillus subtilis (ATCC 6633). 2 An alternative microorganism is Micrococcus luteus (Kocuria rhizophila), ATCC 9341. 3 An alternative to Clostridium sporogenes, when a nonspore-forming microorganism is desired, is Bacetroides vulgatus (ATCC 8482). [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.] 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 Confirm the sterility of each sterilized batch of medium by incubating a portion of the media at the specified incubation temperature for 14 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 microorganisms are indicated in Table 1. Inoculate portions of Fluid Thioglycollate Medium with a small number (not more than 100 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
1

. Suitable strains of

Alternative Fluid Thioglycollate Medium with a small number (not more than 100 cfu) of Clostridium sporogenes. Inoculate portions of SoybeanCasein Digest Medium

with a small number (not more than 100 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 3 days in the case of bacteria and not more than 5 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 1 month, provided that they are tested for growth promotion within 2 weeks of the time of use and that color indicator requirements are met. If stored in tight containers, the media can be used for 1 year, provided that they are tested for growth promotion within 3 months of the time of use and that the color indicator requirements are met.

DILUTING AND RINSING FLUIDS FOR MEMBRANE FILTRATION Fluid A PREPARATION Dissolve 1 g of peptic digest of animal tissue in water to make 1 L, filter or centrifuge to clarify, if necessary, and adjust to a pH of 7.1 0.2. Dispense into containers, and sterilize using a validated process.

PREPARATION FOR PENICILLINS OR CEPHALOSPORINS 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 L of Fluid A add 1 mL of polysorbate 80, adjust to a pH of 7.1 0.2, 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 5.0 g of peptic digest of animal tissue, 3.0 g of beef extract, and 10.0 g of polysorbate 80 in water to make 1 L. Adjust the pH to obtain, after sterilization, a pH of 6.9 0.2. 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 100 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 100 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 5 days. 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 Quantity per Container Liquids (other than anitbiotics) Less than 1 mL 140 mL The whole contents of each container Half the contents of each container, but not less than 1 mL Minimum Quantity to be Used (unless otherwise justified and authorized)

Greater than 40 mL, and not greater than 100 20 mL mL Greater than 100 mL Antibiotic liquids Other preparations soluble in water or in isopropyl myristate 10% of the contents of the container, but not less than 20 mL 1 mL The whole contents of each container to provide not less than 200 mg

Quantity per Container Insoluble preparations, creams, and ointments to be suspended or emulsified Solids Less than 50 mg 50 mg or more, but less than 300 mg 300 mg5 g Greater than 5 g Devices Catgut and other surgical sutures for veterinary use Surgical dressing/cotton/gauze (in packages) Sutures and other individually packaged single-use material Other medical devices

Minimum Quantity to be Used (unless otherwise justified and authorized) Use the contents of each container to provide not less than 200 mg

The whole contents of each container Half the contents of each container, but not less than 50 mg 150 mg 500 mg

3 sections of a strand (each 30-cm long) 100 mg per package The whole device 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) 10% or 4 containers, whichever is the greater 10 containers 2% or 20 containers, whichever is less 2% or 10 containers, whichever is less

Number of Items in the Batch Parenteral preparations Not more than 100 containers More than 100 but not more than 500 containers More than 500 containers For large-volume parenterals Antibiotic solids Pharmacy bulk packages (<5 g)

20 containers

Number of Items in the Batch Pharmacy bulk packages ( 5 g) Bulks and blends

Minimum Number of Items to be Tested for Each Medium (unless otherwise justified and authorized) 6 containers See Bulk solid products

Ophthalmic and other noninjectable preparations Not more than 200 containers More than 200 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 Not more than 100 articles More than 100, but not more than 500 articles More than 500 articles 2 % or 5 packages, whichever is the greater, up to a maximum total of 20 packages 10% or 4 articles, whichever is greater 10 articles 2% or 20 articles, whichever is less 5% or 2 containers, whichever is the greater 10 containers

Bulk solid products Up to 4 containers More than 4 containers, but not more than 50 containers More than 50 containers Each container 20% or 4 containers, whichever is greater 2% or 10 containers, whichever is greater

* If the contents of one container are enough to inoculate the two media, this 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 0.45 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 50 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 (see Diluting and Rinsing Fluids for Membrane Filtration) Fluid A

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. 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 5 times 200 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 14 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 for Membrane Filtration), Fluid A (Diluting and Rinsing Fluids

and proceed with the test as described above for OILS and OILY SOLUTIONS

Aqueous Solutions using a membrane appropriate to the chosen solvent. 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 100 mL of a suitable sterile solution such as Membrane Filtration) Fluid A (see Diluting and Rinsing Fluids for

containing a suitable emulsifying agent at a concentration

shown to be appropriate in the validation of the test, for example polysorbate 80 at a concentration of 10 g per L (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. 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 1% in isopropyl myristate as described above, by heating, if necessary, to not more than 40 . In exceptional cases it may be necessary to heat to not more than 44 . 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 20 containers, aseptically transfer about 300 mg of solids, into a sterile 500-mL conical flask, dissolve in about 200 mL of Fluid A (see Diluting and Rinsing Fluids for Membrane Filtration), and mix; or constitute, as directed in the labeling, each of 20 containers and transfer a quantity of liquid or suspension, equivalent to about 300 mg of solids, into a sterile 500-mL conical flask, dissolve in about 200 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 6 containers, aseptically transfer

about 1 g of solids into a sterile 500-mL conical flask, dissolve in about 200 mL of Fluid A, and mix; or constitute, as directed in the labeling, each of 6 containers and transfer a quantity of liquid, equivalent to about 1 g of solids, into a sterile 500-mL conical flask, dissolve in about 200 mL of Fluid A, and mix. Proceed as directed for Aqueous Solutions. 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 6 g of solids, and transfer to a sterile 500-mL conical flask. Dissolve in about 200 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 20 for about 1 hour. If feasible, allow the propellant to escape before aseptically opening the container, and transfer the contents to a sterile pooling vessel. Add 100 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 10 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 10% 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 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 80 at a concentration of 10 g per L. OINTMENTS and CREAMS Prepare by diluting to about 1 in 10 by emulsifying with the chosen emulsifying agent in a suitable sterile diluent such as Membrane Filtration). emulsifying agent. Incubate the inoculated media for not less than 14 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 30-cm long, which have been cut off from the beginning, the center, and the Fluid A (see Diluting and Rinsing Fluids for

Transfer the diluted product to a medium not containing an

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 (20 mL to 150 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 200 mL of Fluid Thioglycollate Medium, and mix. Similarly, transfer the same quantity to 200 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 100- to 500-mg each from the innermost part 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, 14 days after the beginning of incubation transfer

portions (each not less than 1 mL) of the medium to fresh vessels of the same medium, and then incubate the original and transfer vessels for not less than 4 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. 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 100 mL with a suitable sterile solution, such as Fluid A (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. 1 In appropriate cases, periodic testing of the different batches prepared from the same lot of dehydrated medium is acceptable. Auxiliary Information Staff Liaison : Radhakrishna S Tirumalai, Scientist Expert Committee : (MSA05) Microbiology and Sterility Assurance USP29NF24 Page 2508 Phone Number : 1-301-816-8339