It is established to ensure the Sterility test for Parenterals.
It is applicable in the microbiology department of the Quality Control laboratory.
Quality Control Manager
4. Introduction About Sterility test for Parenterals
The following procedures are applicable for determining whether an article purporting to be sterile complies with the test for sterility. 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.
These 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 methods, the result so obtained is conclusive evidence of the failure of the article to meet the requirements of the test for sterility, even if a different result is obtained by an alternative procedure.
5. Sterility test for Parenterals Requirements
- Screwed test tubes (100ml)
- Sterility oven
- Laminar flow
- Measuring cylinders
- Conical flasks (1L)
- Surgical Cotton
- Aluminum Foil
- Weighing balance
- Heating plate with stirring arrangement
- Filtration assembly
- Membrane filter (0.45mm pore size, 47mm dia, sterilized)
- Isopropyl alcohol
- Distill Water
- Fluid thioglycolate medium
- Tryptone soya broth
5.3 Testing Facilities
The following two types of facilities are used for the Sterility test for Parenterals.
5.3.1 Clean Rooms and Clean Zones
A cleanroom of a sterility testing facility is maintained under microbiological control criteria appropriate for the critical zones in an aseptic processing facility. When a clean zone is used for sterility testing, it must also meet the same microbiological control criteria.
When performing the sterility test in the isolator, the article meets the requirements of the test for the sterility when no microbial growth is observed. When microbial growth is observed and confirmed microscopically, the article does not meet the requirements of the test. If the microbial growth can be without a doubt ascribed to a loss of physical integrity of the isolator or to gross contamination due to faulty techniques or unsterile materials within the isolator enclosure, the test is invalid. Repeat the test as indicated below.
5.5 PREPARATION OF 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. Media are sterilized in autoclaves using a validated process.
Fluid Thioglycollate Medium
|Agar, granulated (moisture content
|Not exceeding 15%)
|Yeast Extract (water-soluble)
|Pancreatic Digest of Casein
|or Thioglycolic Acid
|Resazurin Sodium Solution
|(1 in 1000), freshly prepared)
Mix and heat until a solution is affected. Adjust the pH of the solution with 1 N sodium hydroxide so that after sterilization it will have a pH of 7.1 ± 0.2. Filter while hot through a filter paper, if necessary. Transfer the medium to suitable containers that provide a ratio of surface to a 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, and sterilize as directed above. If more than the upper one-third of the medium has a pink color, the medium may be restored once by heating the containers until the pink color disappears. When ready for use, not more than the upper one-third of the medium in a container should have a pink color. Incubate under aerobic conditions.
Tryptone soya broth
|Pancreatic Digest of Casein
|Papaic Digest of Soybean Meal
|Dibasic Potassium Phosphate
Dissolve the solids in the 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, and dispense into suitable containers. Sterilize as directed above or by a validated filtration process. Incubate under aerobic conditions.
5.6 STERILITY OF MEDIA
Confirm the sterility of each sterilized batch of the medium by incubating a portion of the batch at the specified incubation temperature for not less than 14 days or by incubating uninoculated containers as negative controls during a sterility test procedure. This will serve as a Medium control.
5.7 GROWTH PROMOTION TEST
Each lot of dehydrated medium bearing the manufacturer’s identifying number or each lot of medium prepared from basic ingredients must be tested for its growth-promoting qualities. Separately inoculate, in duplicate, containers of each medium with less than 100 viable microorganisms of each of the strains listed in Table 1, and incubate according to the conditions specified. The test media are satisfactory if visual evidence of growth appears in all inoculated media containers within 5 days of incubation. This test can be conducted simultaneously with the use of the media for sterility test purposes. This will serve as culture control. However, the sterility test is considered invalid if the sterility of the media or this growth promotion test is not successful.
Ready-to-Use Media—Commercially prepared media stored in tight containers may be used provided that the requirements of the Sterility of Media and the Growth Promotion Test are met.
5.9 DILUTING AND RINSING FLUIDS FOR MEMBRANE FILTRATION
5.9.1 Fluid A
Preparation — Dissolve 1 g of peptic digest of animal tissue in water to make 1 liter, 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.
5.9.2 Fluid D
To each liter 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.
5.9.3 Fluid K
Dissolve 5.0 g of peptic digest of animal tissue, 3.0 g of beef extract, and 80 g of polysorbate 80 in water to make 1 liter. Adjust the pH to obtain, after sterilization, a pH of 6.9 ± 0.2. Dispense into containers, and sterilize using a validated process.
5.10 VALIDATION TESTS FOR BACTERIOSTASIS AND FUNGISTATIC
Before instituting the use of a sterility test procedure for an article, ensure that any bacteriostasis and fungistatic activity inherent in the article to be tested does not adversely affect the reliability of the test and that the test procedure to be instituted is otherwise suitable for use with the article. Prepare to dilute cultures of bacteria and fungi from the strains of microorganisms listed in Table 1 to obtain a final concentration of microorganisms in the product of less than 100 CFU per mL. Repeat the test method for each microorganism used. [NOTE—If the procedure or media specified under Method I does not eliminate the antimicrobial activity, alternative media or neutralizers can be used as long as they are capable of overcoming bacteriostasis or fungistatic.]
5.10.1 Method I
Procedure — Method I is used for the validation of bacteriostasis and fungistatic by the membrane filtration method. Filter the specified quantity of the test specimen; using the same number of containers per single filter unit or canister as will be used in the sterility test. If necessary, rinse the membrane with a minimum of three 100-mL portions of the appropriate rinsing fluid. Inoculate the final rinse with less than 100 CFU. Repeat the rinse procedure on another filter that has not been exposed to the specimen under test. This filter will serve as the positive control. Place the filter or filter halves into the specified test medium, or add the specified medium to the canister containing the membrane filter. Repeat the procedure for the appropriate microorganisms and media specified in Table 1, and incubate the containers at the appropriate temperature for not more than 7 days.
If the growth of each test organism in the test containers is visually comparable to the growth in the positive control, use the same amounts of the article, number, and volume of rinses, and medium when conducting the sterility test. If the growth of the test organisms in the test containers is not visually comparable to that in the positive control, the amount of article used is bacteriostasis or fungistasis. Repeat the test, using a larger number of rinses. Changes in the type of membrane filter used and in the use of neutralizing agents, if available, may reduce the antimicrobial effect of the article (see Interpretation under Method II). If five rinses, each of about 500 mL, fail to neutralize the antimicrobial residue on the test filter membrane, proceed with the sterility test.
5.10.3 Method II
Procedure — Method II is used for the validation of bacteriostasis and fungistatic by the direct transfer method. Inoculate two containers of each sterility test medium with less than 100 colony-forming units, using the volume of medium for each appropriate microorganism specified in Table 1. Add the specified portion of the article under test to one of the inoculated containers of each medium. The other inoculated container is a positive control. Repeat the procedure for each appropriate microorganism, and incubate the containers at the appropriate temperature for not more than 7 days.
If the growth of the test organisms in the test container is not visually comparable to that of the inoculated control container, the article is bacteriostasis or fungistatic. The use of a sterile neutralizing agent, such as polysorbate 80, lecithin, azolectin, or b-lactamase, may be appropriate. If a neutralizing agent is not effective, establish suitable increased volumes of the medium. Use the smallest volume of medium in which the growth of test microorganisms in the presence of the article is not adversely affected. [NOTE—If the medium volume is increased to 2000 mL and antimicrobial activity is still present, proceed with the sterility test using the 2000 mL of medium.] Volumes of medium greater than 2000 mL may be needed for testing medical devices, to permit complete immersion of the device.
6. General Procedure for Sterility test for Parenterals
6.1 Sample Preparation
6.1.1 Number of Articles to Be Tested
Test the number of articles specified in Table 2.
6.1.2 Opening Articles
Great care must be exercised when opening an article so that the sample to be tested for sterility is not contaminated by microorganisms present on the exterior of the container. The exterior surfaces of ampoules and closures of vials and bottles must be cleaned with a suitable decontaminating agent, and the containers must be placed in an environment that prevents recontamination of the exterior surfaces. If the vial contents are packaged under vacuum, admit sterile air by means of a suitable sterile device, such as a needle attached to a membrane filter holder containing a sterilizing grade filter. For articles such as purified cotton, gauze, surgical dressing, sutures, and related articles, decontaminate the outer package, and open the package or container aseptically.
6.1.3 Quantity of Article
When using the Membrane Filtration Method, use the entire contents of each container.
6.1.4 Volume of medium
Take about 60ml of the medium in 100ml sterilized screwed test tube.
6.1.5 Incubation Conditions
Incubate for not less than 14 days at 32.5 ± 2.5° for the Fluid Thioglycollate Medium or at 22.5 ± 2.5° for the tryptone soya broth. Observe the tubes of media on a periodic basis over the 14 days of incubation. If the test specimen is positive before 14 days of incubation, further incubation is not necessary. For products terminally sterilized by a validated moist heat process, incubate the test specimen for not less than 7 days, if the Membrane Filtration Method is used.
6.2 Test Procedure for Sterility test for Parenterals
6.2.1 Membrane Filtration Method
A suitable membrane filter unit consists of an assembly that facilitates the aseptic handling of the test articles and allows the processed membrane to be removed aseptically for transfer to appropriate media or an assembly where sterile media can be added to the sealed filter and the membrane incubated in situ. The filter units and the membranes must be sterilized and stored in a manner that maintains the performance characteristics of the filter and ensures that the filter and the assembly remain sterile. When the article to be tested is oil, the membrane, and the filter assembly must be thoroughly dried before use. Agitate the container and aseptically transfer the content of the total number of specimens tested either directly into one or more separate membrane filter units or to separate pooling vessels prior to transfer. Immediately pass each specimen through the filter with the aid of vacuum or pressure. If only one filter unit is used, aseptically remove the membrane from the holder, cut the membrane in half, and immerse half of the membrane in each of the specified media. If two or more filter units are used, place an equal number or portions of filter membrane in each of the specified media. During all manipulations, avoid excessive aeration of the Fluid Thioglycollate Medium. If the substance under test is a viscous liquid or suspension and is not adaptable to rapid filtration, aseptically add a sufficient quantity of the appropriate diluting fluid to the pooled specimen prior to filtration to increase the flow rate.
6.2.2 Direct Transfer Method
Non-filterable Liquids — Agitate the containers and aseptically withdraw, from a sufficient number of units, the volumes for each medium, as indicated in Table 2. Mix each test specimen with the appropriate medium, but do not aerate excessively. Proceed as directed under Procedure.
6.2.3 Results Interpretation of Sterility test for Parenterals.
The article meets the requirements of the test for sterility when no microbial growth is observed. When microbial growth is observed and confirmed microscopically, the article does not meet the requirements for sterility. However, if the microbial growth can be without a doubt ascribed to faulty aseptic techniques or materials used in conducting the sterility testing procedures, the test is invalid and must be repeated. Daily observation of the microbial growth will be observed and recorded on Performa.
Table 1. Test Microorganisms Suitable for Use in the Growth Promotion Test during Sterility test for Parenterals.
|Incubation (7 days)
|32.5 ± 2.5°
|Pseudomonas aeruginosa 3
|32.5 ± 2.5°
|Clostridium sporogenes 4
|32.5 ± 2.5°
|Tryptic soya broth
|22.5 ± 2.5°
|22.5 ± 2.5°
|22.5 ± 2.5°
2 An alternative to Staphylococcus aureus is Bacillus subtilis (ATCC 6633).
3 An alternative microorganism is Micrococcus luteus, (ATCC No. 9341).
Table 2. Minimum Number of Articles to Be Tested in Relation to the Number of Articles in the Batch for Sterility test for Parenterals.
|Number of Articles in the Batch
Injections / for Injections
|Number of Articles to Be Tested
|10% or 4 articles, whichever is greater
|2% or 20 articles, whichever is less
|2% or 10 containers, whichever is less