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Guidelines for sterility testing of therapeutic goods

13 September 2006

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4. Test methods

400. Relevant sections  of the BP/Ph Eur: 'Test for sterility of the  product  to be examined'; 'Validation test'; 'Observation and interpretation of results'; 'Application of the test to parenteral preparations, ophthalmic and other non-injectable preparations required to comply with the test for sterility'

General methodology

401. Tests for sterility are carried out by the method of Membrane Filtration, by the method of Direct Transfer or by Addition of Concentrated Medium to the product. The method of Membrane Filtration should be used as the method of choice wherever feasible.

402. Fluid Thioglycollate Medium (Medium 1) and Soybean-Casein Digest Medium (Medium 2) are the media generally used for tests for sterility (see Section 6). Alternative media types may be appropriate where the nature of the product or method of manufacture can result in the presence of fastidious organisms (eg vaccines, blood products). Validation studies should indicate that alternative media are capable of supporting the growth of a wide range of micro- organisms in the presence of the product.

403. Where the preparation to be tested has antimicrobial effects, these effects may be reduced or neutralised by adding an appropriate substance to the specified test media, to diluents or solvents, or to the preparation prior to testing. Media so modified should be subjected to the tests described for unmodified media in clauses 608-616 and should only be used in tests for sterility if found to comply.

404. Containers of Medium 1 are incubated at 30 - 35°C and Medium 2 at 20 - 25°C.

Test method validation

405. Before tests for sterility for any product are initially carried out, it is necessary to demonstrate the validity of the test method used by recovery of a small number of micro- organisms in the presence of the product (see Annex I Guidance on Obtaining Small Numbers of Vegetative Organisms and Spores). It is preferable to add these challenge organisms directly to the product prior to membrane filtration or direct inoculation; where this is not practicable due to inhibition or irreversible binding by the product, the challenge organisms should be added to the last rinse solution if the membrane filtration method is used, or directly to the media containing the product if the direct method is used.

406. Validation should mimic the test proper in every detail, such as in the volumes of media used, quantities and dilutions of product and diluents: the approach depends on the method of test and details are given in each section. It may be performed concurrently with the actual test for sterility but should be confirmed as successful before the results of the sterility test are interpreted.

407. Validation is to be performed when the test for sterility has to be carried out on reformulated or new product, or whenever there is a change in the experimental conditions of the test. It is good practice to revalidate test methodology every 12 months although this is not a pharmacopoeial requirement and the frequency can be varied depending on frequency of manufacture, the nature and ingredients of the product and the frequency of stasis testing.

408. If a test method cannot be satisfactorily validated the regulatory authority should be notified.

409. All validation procedures should be carried out by personnel who are responsible for the routine testing of the product and should be done for each facility manufacturing that product.

Method of membrane filtration

Procedures

410. The filter should be a membrane filter disc of cellulose esters or other suitable plastics, having a nominal average pore diameter not exceeding 0.45µm. The membrane should be held firmly in a filtration unit which consists of a supporting base for the membrane, a receptacle for the fluid to be tested, a collecting reservoir for the filtered fluid, and the necessary tubes or connections. The apparatus is so designed that the solution to be filtered can be introduced and filtered under aseptic conditions. It permits the aseptic removal of the membrane for transfer to medium or it is suitable for carrying out the incubation after adding the medium to the apparatus itself.

411. Where the product has antimicrobial activity, the use of hydrophobic edged membranes is recommended so as to facilitate washing, unless self-contained canister systems are employed.

412. Cellulose nitrate filters are recommended for aqueous, oily and weakly alcoholic solutions and cellulose acetate filters for strongly alcoholic solutions.

413. The entire unit should be sterilised by appropriate means with the membrane filter and sterile airways in place. The method of sterilisation should not be deleterious to the membrane, eg, weaken it or change the nominal average pore diameter. The sterile airways should provide free access to the sterilising agent. After sterilisation, the apparatus should be free of leaks to the atmosphere except through the sterile airways.

414. The filter should be pre-wetted with diluent or solvent before filtration to minimise the retention of sample, particularly where small volumes and antibiotics are tested. A visual check of the integrity of the filter membrane should be carried out after filtration has been completed and the test shall be invalid if defects are apparent.

415. The specific diluents referred to in Table 4 and in the sections below are not obligatory and alternatives may be used provided they are compatible with the membrane and do not have antimicrobial activity as demonstrated by validation studies. It is assumed that membranes of approximately 50 mm diameter are used in the methods described below. If filters of a different diameter are used the volumes of diluents and wash solutions should be adjusted appropriately. The total volume washed through one single membrane should not exceed 1000 mL unless otherwise justified and authorised.

Aqueous solutions and suspensions which may be filtered directly

416. The prescribed volumes (Tables 2 and 3) of aqueous solutions and suspensions which can be filtered without prior treatment or dilution are transferred from the containers of the product to a sterile filtration unit. Filtration is then carried out with the aid of suction or pressure.

417. Without delay the filter membrane(s) should be washed not less than three times with the diluent specified in Table 4 or an equivalent. Throughout the operation the membrane should remain covered with liquid. If the original preparation contains a preservative or has inherent antimicrobial activity, additional washes may be needed and/or the diluent may include an antimicrobial inactivator. The volume of diluent must be equal to that used during validation.

418. After filtration and washing, aseptically divide the filter into two parts of approximately equal surface area and transfer one part to Medium 1 and the other part to Medium 2.

419. The volume of Medium 1 should be such that the air space above the medium in the container is minimised. The volume of Medium 2 should be such that sufficient air space is left above the  medium to provide conditions that permit the  growth of obligate aerobes. This condition applies irrespective of the filtration system used.

Table 4: Sample treatments, solvents and diluents suggested for use in sterility tests using the method of membrane filtration1
Class of Sample Treatment before filtration (solution or dilution)3 Diluent for washing after filtration3
Aqueous solutions Containing lecithin - Diluent 2
Others - Diluent 1 or 2
With ingredient containing beta-lactam ring Diluent 3 (one or more of the washes)
Water soluble substances Containing lecithin Dissolve in sterile water or suitable solvent Diluent 2
Others Dissolve in sterile water or suitable solvent Diluent 1 or 2
With ingredient containing beta-lactam ring Dissolve in sterile water or suitable solvent or Diluent 3 Diluent 3 (one or more of the washes)
Water insoluble substances (in solid form or as aqueous suspension) Containing lecithin Suitable solvent Diluent 2
Not containing lecithin Suitable solvent Diluent 1 or 2
Containing a beta-lactam antibiotic Diluent 3 or other solvent containing penicillinase Diluent 3
Ointments and oily preparations Dissolve in suitable solvent2 Diluent 2
  1. In every case an equally or more effective diluent may be substituted for that suggested in the table.
  2. In some cases it may be necessary to use a mixture of solvents or several different solvents in succession. Where the sample contains an antibiotic with a beta-lactam ring it may be necessary to add penicillinase to one of the solvents or to the solvent mixture.
  3. Refer to Annex III for composition and preparation of diluents.

420. If test apparatus is used in which medium is added to the apparatus and the membrane incubated in situ,the sample should be divided between two units or multiples thereof. Add Medium 1 to one of the units and Medium 2 to the other unit.

421. The minimum test is one in which a single membrane is divided into two parts and one part is inoculated into Medium 1 and one part into Medium 2. The number of containers tested may be increased to increase the statistical information provided by a test. If the full sample cannot be passed through a single membrane because of filtration difficulties or unacceptable levels of residual antimicrobial substance(s) in the filter membrane, the sample may be divided into portions and each filtered separately. However, the transfer of membranes or parts of membranes into the two media should not differ from the above proportions. Incubate test vessels of Medium 1 at 30 - 35°C and the vessels of Medium 2 at 20 - 25°C.

Aqueous solutions and suspensions which must be diluted or treated prior to filtration

422. Where aqueous solutions or suspensions must be diluted or treated prior to filtration they should be diluted with diluents specified in Table 4 or a suitable alternative sterile diluent or solvent and should be filtered, washed or otherwise treated as for those which may be filtered directly.

423. 'Suitable alternative' is any other sterilised diluent in which the suspended substance is soluble or which enables the substance to pass through the filter. Such alternative diluents should not exhibit antimicrobial activity as demonstrated by validation studies.

424. The diluted or treated preparation should be filtered as described above in clauses 416-421.

Soluble or dispersible solids

425. Prior to filtration, the quantity of a solid to be tested (see Tables 2 and 3), is transferred from each container to one or more vessels to be pooled, dissolved or otherwise treated as permitted by this method. This should be done in one or more vessels containing the suitable solvent.

426. In the case of solids in final dosage form, measured volumes of the suitable solvent may be added directly to the final containers, and the test sample may then be withdrawn in the form of a solution or a suspension.

427. Appropriate diluents for use in these operations are listed in Table 4. A 'suitable solvent' for dissolving a water-insoluble solid is a solvent or a liquid that facilitates dissolution of the solid and its passage through the filters. It should be sterile and should not exhibit antimicrobial activity or change the nominal pore diameter of the filter membrane in the conditions of the test as demonstrated by validation studies.

428. The resulting preparation should be filtered as described above in clauses 416-421.

Ointments and oily preparations

429. Oils and oily solutions of sufficiently low viscosity may be filtered without  dilution through a dry membrane.

430. 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.

431. Allow the oil to penetrate the membrane by its own weight, then filter, applying the pressure or suction gradually. Wash the membrane not less than three times by filtering through it about 100 mL of a suitable sterile solution such as Diluent 2.

432. Ointments in a fatty base and emulsions of the water-in-oil type may be diluted to 1 per cent in isopropyl myristate at a temperature of 40°C, but not more than 44°C. As rapidly as possible the preparation should be filtered and the membranes washed without delay, as described above for oils and oily solutions.

433. The resulting preparation and filters should be cultured as described above in clauses 418-421.

Table 5: Micro-organisms for use in growth promotion, validation and stasis tests1
Micro-organism Incubation Conditions
Species Suitable strain Temperature (°C) Maximum duration

Type: anaerobic bacteria

30 – 35

3 days for growth promotion.

5 days for validation and stasis.

Clostridium sporogenes ATCC 19404
CIP 79.3
NCTC 532
ATCC 11437

Type: aerobic bacteria

30 – 35

3 days for growth promotion.

5 days for validation and stasis.

Staphylococcus aureus ATCC 6538
CIP 4.83
NCTC 10788
NCIMB 9518

Pseudomonas aeruginosa ATCC 9027
NCIMB 8626
CIP 82.118

Bacillus subtilis ATCC 6633
CIP 52.62
NCIMB 8054

20 – 25

Type: fungi

20 – 25 5 days
Candida albicans ATCC 10231
IP 48.72
NCPF 3179

Aspergillus niger ATCC 16404
IP 1431.83
IMI 149007

  1. This Table incorporates Table 2.6.1.-1 (BP/Ph Eur) and Table 1 (USP).

Initial validation of the test method - testing for residual antimicrobial activity

434. To validate the test method, carry out the test procedures as described in the relevant section above, up to the final wash procedure. To the final wash add an inoculum of not more than 100 viable cells of each of the specified aerobic bacteria, anaerobic bacteria and fungi.. (For guidance on preparation of inocula, see Annex I Guidance on Obtaining Small Numbers of Vegetative Organisms and Spores).

435. Add Clostridium sporogenes ATCC 19404, Staphylococcus aureus ATCC 6538 and Pseudomonas aeruginosa ATCC 9027 to Medium 1 and Candida albicans ATCC 10231, Bacillus subtilis ATCC 6633 and Aspergillus niger ATCC 16404 to Medium 2. Other appropriate strains of challenge organisms are listed in Table 5..

436. After the final wash with the added micro-organisms has been passed through the filter, incubate one filter disc in Medium 1 at 30 - 35°C and one in Medium 2 at 20 - 25°C.

437. If different culture conditions are to be used in the test for justified reasons, these must be validated using challenge organisms appropriate for the conditions.

438. Periodically, strains of micro-organisms collected from the manufacturing environment should be used as challenge organisms.

439. Growth of each of the added micro-organisms should be apparent within 48 hours. If conspicuous growth does not occur within 5 days for each bacteria and fungi the test procedure is not valid and must be modified (e.g. by using additional washes, using antagonists to the antimicrobial agent or other procedure) until conspicuous growth does occur when tests as above are carried out.

440. If the membrane is found to be free of such antimicrobial activity when first tested or after modification of procedures, application of the test to every sample is not necessary. (See also clause 407).

Method of direct transfer

Procedures

Liquids and soluble or dispersible solids

441. Transfer the quantity of the preparation to be examined as indicated in Table 3 directly into Medium 1 and Medium 2. Approximately equal quantities of the preparation should be added to each vessel of medium. Incubate the test vessels of Medium 1 at 30 - 35°C and the vessels of Medium 2 at 20 - 25°C.

442. The volume of Medium 1 should be such that the air space above the medium in the container is minimised. The volume of Medium 2 should be such that sufficient air space is left above the medium to provide conditions that permit the growth of obligate aerobes.

443. Unless otherwise prescribed, in no case should the volume of material under test be greater than 10% of the volume of the medium alone, ie, 90% medium and 10% product.

444. In the case of soluble or dispersible solids, a measured volume of Purified Water, or a suitable sterilised diluent or solvent which does not manifest antimicrobial activity as demonstrated by validation studies, should be added to each container of the solid. After the contents have been dissolved or dispersed, the specified quantity of the product should then be added in the form of a solution or suspension to the test media. Alternatively, the solid material may be transferred directly to the test media.

445. If a large volume of product is to be tested it may be preferable to use concentrated media, prepared so as to take the subsequent dilution into account. Where appropriate the concentrated medium may be added directly to the product in its container.

446. Any additional diluents, solvents or procedures for carrying out the test should be validated.

Solid articles

447. Wherever possible solid articles such as devices should be tested by immersion in or filling with culture media.

448. Aseptically dismantle all articles as completely as possible. Articles such as tubing may need to be cut. Other articles may need to be broken into smaller parts to allow access of medium to all surfaces of the article.

449. Immerse all parts of each article in sufficient medium contained in one vessel to completely cover all parts. The volume of Medium 1 should be such that the air space above the medium in the container is minimised. The volume of Medium 2 should be such that sufficient air space is left above the medium to provide conditions that permit the growth of obligate aerobes.

450. Place half the articles into Medium 1 and the remaining half into Medium 2. Incubate the test vessels of Medium 1 at 30 - 35°C and the vessels of Medium 2 at 20 - 25°C.

451. Where the product is a dressing, the whole article need not be tested. As a minimum, portions of 100-500 mg should be cut from that part of the dressing that is most inaccessible to sterilant. Articles may be pooled.

452. If the size of an article is such that all its parts are not covered by 2000 mL of medium, then those parts likely to be most easily accessible to the sterilant may be omitted. Unless the product is a dressing, before a test is adopted which omits parts of an article from routine testing, the proposed procedure should be discussed with the competent authority. Parts of an article should not be omitted from testing in order for articles to be pooled.

453. Alternatively, a larger vessel containing additional medium and capable of accommodating all parts of the article may be used.

454. Alternatively, if a large article cannot readily be cut into pieces, or only the fluid pathway of the device is intended to be sterile, medium should be added to the article aseptically and it should then be sealed and incubated.

455. If none of the above methods is practicable the article may be rinsed three times with suitable volumes of medium, so that all surfaces of the article which are required to be sterile come into intimate contact with medium. The entire washings from each article are then tested by the method of Membrane Filtration. This method is not as sensitive as those described above because micro-organisms adhering to surfaces may not be removed by washing. It should be used only as a last resort.

456. Care should be taken to ensure that entrapped air does not prevent the medium from making contact with all parts of the internal surfaces of an article. To facilitate this contact a surfactant agent is included in Medium 1 and Medium 2; Medium 1 may also be modified by the omission of agar.

Ointments and oily preparations

457. Ointments and oily preparations may be tested by the method of Direct Transfer if testing by the method of Membrane Filtration is not feasible, i.e. when a suitable solvent is not available (see clauses 429-430).

458. Before addition to media, ointments and creams may be diluted approximately 1 in 10 by emulsifying with a suitable emulsifying agent in a suitable sterile diluent to improve contact between the sample and the medium (polysorbate 80 or light liquid paraffin may be useful). In this case it may be appropriate to use Medium 1 and Medium 2 without polysorbate 80.

459. For oily liquids media containing an emulsifying agent should be used. Polysorbate 80 at 10 g/L, (p-tert-octylphenoxy) polyoxyethanol at 1 g/L, or other emulsifying agents in appropriate concentration may be suitable.

Initial validation of the test method - testing for antimicrobial activity

460. The goods to be tested for sterility should be tested for antimicrobial activity during the product development stages, if this is possible. If they are found to have such activity, preparatory or test procedures will need to be modified to neutralise this activity.

461. If goods are found to be free of such activity when first tested, or after modification of procedures, application of the test for antimicrobial activity to every sample is not necessary. (See also clauses 405-409).

462. To demonstrate that the mixture does not manifest antimicrobial activity carry out the test as described above up to the incubation step and add an inoculum of viable cells of the specified aerobic bacteria, anaerobic bacteria and fungi.

463. To one vessel containing the test sample in Medium 1, add an inoculum of Clostridium sporogenes ATCC 19404, Staphylococcus aureus ATCC 6538 and Pseudomonas aeruginosa ATCC 9027 and incubate at 30 – 35°C. To a second vessel containing the test sample in Medium 2 add an inoculum of Candida albicans ATCC 10231, Bacillus subtilis ATCC 6633 and Aspergillus niger ATCC 16404 to Medium 2 and incubate the vessel at 20 – 25°C.

464. Other appropriate strains of challenge organisms are listed in Table 5.

465. In each case the number of micro-organisms in the inoculum is to be not more than 100 CFU. (For guidance on preparation of inocula, see Annex I Guidance on Obtaining Small Numbers of Vegetative Organisms and Spores).

466. Growth of each of the added micro-organisms should be apparent within 48 hours. If conspicuous growth does not occur within 5 days, the test procedure is not valid and must be modified (e.g. by using additional washes, using antagonists to the antimicrobial agent or other procedure) until conspicuous growth does occur when tests as above are carried out.

467. Periodically, the strains referred to above should be supplemented by strains of micro- organisms collected from the manufacturing environment.

Negative product control tests

468. The results of negative product control tests facilitate the interpretation of sterility test results, particularly when used to declare a test invalid because of contamination in the negative product controls.

469. During each working session (i.e. that uninterrupted period of time in which a sample or group of samples is tested) in which sterility testing is carried out, at least ten negative product control containers should be tested. For a direct inoculation test these controls should be tested where possible at regular intervals during the test session.

470. A negative product control is usually a terminally sterilised item of undoubted sterility, that is, it has been subjected to the equivalent of two sterilisation cycles by autoclaving or by dry heat sterilisation, or 50 kGy of gamma irradiation. Acceptable alternatives could be a container that has been aseptically filled and then subjected to 25 kGy or a container of medium that has been filled during a media fill validation, incubated for 14 days and been found to be sterile.

471. A negative control should be similar in type and container (or packaging if a device) to the product under test. The essential element of the negative control is that the manipulations involved in testing the control should be similar to those involved in testing the product. There should be similar risks of introducing contamination in the control and product tests.

472. A suitable negative product control for an aqueous product could be distilled water in a similar container. A negative product control for testing an ointment could be a container of liquid paraffin or ointment base that has been sterilised by dry heat; pouring the liquid paraffin from a container would be adequate to simulate squeezing of ointment from a tube. For disposable devices, a section of glass or plastic tubing packaged in a manner similar to the device, and sterilised by gamma irradiation, could serve as a negative control.

473. Where a retest is being carried out in the working session these simulated negative controls should be processed concurrently with that retest.

474. The negative control contamination rate should be calculated and recorded. In order to derive the maximum information from the results of sterility tests it is essential that the level of contamination detected in negative control tests be minimal.

Incubation and examination of sterility tests

475. Incubate all test vessels of Medium 1 (or equivalent medium - see clause 402) at 30 - 35°C. Incubate the vessels of Medium 2 (or equivalent medium - see clause 402) at 20 - 25°C.

476. All vessels should bear the identity of the product or control being tested, the medium used, the temperature of incubation and date of inoculation.

477. All test and control vessels, other than the subcultured vessels referred to below, must be incubated for at least 14 days unless microbial contamination is detected at an earlier time.

478. At intervals during the incubation period examine each vessel for evidence of microbial growth; a suitable interval is 2 working days. Care should be taken to prevent undue agitation of the Medium 1. At the end of the incubation period examine each vessel again for evidence of microbial growth after agitating, swirling or inverting the contents.

479. Preparations that produce a suspension, flocculation or deposit so that the presence or absence of microbial growth cannot be readily seen should be mixed by gentle swirling or inversion at each examination until subcultured. Care should be taken to prevent undue agitation of the Medium 1 and to ensure that anaerobic conditions are maintained as indicated by the resazurin indicator. After 14 days incubation transfer a suitable portion (not less than 1 mL) of the contents to a fresh vessel of the same medium. Incubate the subcultured vessels for not less than 4 days at the same temperature as that at which the original vessel  was incubated. Continue incubation of the original and the subcultured vessels for a total of not less than 14 + 4 days from the original inoculation.

480. If turbidity, precipitate, or other evidence of microbial growth during incubation is seen:

  • examine the suspected growth microscopically by Gram stain;
  • attempt to grow single colonies using appropriate microbiological methods;
  • examine  colonies  of  each  type  of  micro-organism  present  for  their  colonial morphology and cellular morphology by Gram stain;
  • attempt to identify the isolates, as far as the genus, and preferably species.

NOTE: If the identity of isolates is to be used as the basis for invalidating a test, a sensitive method of identification such as molecular typing techniques using RNA/DNA homology is required (see also clause 495).

481. Keep records of these cultures in order to detect a pattern of recurring contaminants in the product. It is recommended that cultures of recurring contaminants be maintained in pure form and used as reference organisms for evaluation of environmental background contamination.

482. Automated or semi-automated biochemical organism identification systems should be subjected to periodic verification using reference strains of organisms that can be traced to a recognised reference culture collection, such as the American Type Culture Collection (ATCC), Maryland, USA, or the National Collection of Type Cultures (NCTC), London, UK.

Monitoring the efficacy of test media at the end of the incubation period (stasis test)

483. The stasis test is not mandated by the Pharmacopoeias but is recommended as part of Good Laboratory Practice (GLP) in relation to method validation and a quality system based on ISO 17025. It is particularly important for antibiotics, slow-release sterile products and for direct inoculation methods where validity of the test depends on the use of an exact amount of product (ie, marginal methodology).

484. The stasis test is intended to demonstrate that the media inoculated with the test preparation will support growth for the full incubation period. For example, it is necessary to show that anaerobiosis is maintained in the Medium 1 to allow the late development of slow- growing anaerobes. It is also necessary to demonstrate that growth promoting qualities of media are retained and that preservative inhibitors remain stable for the full test period.

485. The stasis test will be included in referee testing (see clauses 101 and 105).

486. After incubation of the media has been completed in accordance with the instructions given in clauses 475-479:

  • add to representative vessels containing Medium 1 that has been incubated at 30 – 35°C, an inoculum of viable spores of an anaerobic bacterium eg, Clostridium sporogenes ATCC 19404;
  • add to representative vessels containing Medium 2 that has been incubated at 20 – 25°C an inoculum of viable cells of a fungus, eg, Candida albicans ATCC 10231;

NOTE: Acceptable challenge organisms are listed in Table 5. The list is not exclusive; other micro-organisms may be suitable.

487. In each case the number of organisms in the inoculum is to be not more than 100 CFU (see Annex I Guidance on Obtaining Small Numbers of Vegetative Organisms and Spores).

488. The vessels are returned to their previous temperature and incubation continued. The containers of product should all show growth of the added  organisms within 48  hours. If conspicuous growth is not apparent within 5 days for both bacteria  and fungi the test is considered invalid. Invalid stasis tests may be repeated once. If conspicuous growth is not obtained at the second attempt the test method should be modified and revalidated.

489. If the media are found to support growth of the test micro-organisms then this test need not be applied to every sample. It should be repeated periodically on the relevant categories of products or when product is reformulated. Every 12 months is recommended.

490. Periodically the strains referred to above may be supplemented with appropriate strains of micro-organisms collected from the manufacturing environment.

Interpretation of the test results

491. If microbial growth is not evident in any of the vessels inoculated with the product, the sample tested complies with the test for sterility, provided that growth of challenge organisms has been demonstrated in the stasis test (if performed), in growth promotion tests on the batches of media used and in test method validation. This interpretation applies even if growth occurs in negative product control vessels.

492. If microbial growth is evident the product 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 being examined.

493. If microbial growth is evident, the criteria for invalidating the test are:

  1. the data of the microbiological monitoring of the sterility testing facility show a fault;
  2. a review of the testing procedure used during the test in question reveals a fault;
  3. microbial growth is found in the negative product controls;
  4. after determination of the identity of the micro-organisms 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 technique used in conducting the sterility test procedure.

494. When conditions (a), (b) or (c) apply, the test should be aborted prior to the completion of the incubation period.

495. If condition (d) is to be used as the sole criterion for invalidating a sterility test, it is necessary to employ sensitive typing techniques to demonstrate that a microorganism isolated from the product test is identical to a microorganism isolated from the materials and/or the environment. While routine biochemical/phenotypical identification techniques can demonstrate that two isolates are not identical, these methods are not sufficiently sensitive or reliable enough to provide unequivocal evidence that two isolates are from the same source. Suitably sensitive tests (for example, molecular typing with RNA/DNA homology) are those accepted by microbiologists conducting epidemiological studies to determine that microorganisms are clonally related and have a common origin. Repeat testing based on the biochemical or phenotypical characterisation of environmental and/or product isolates should not be permitted. The test environment can be contaminated by actual product samples, which may contain multiple micro-organisms that are difficult to speciate without employing sensitive typing techniques.

496. If the test is declared to be invalid it may be repeated with the same number of units as in the original test.

497. If there is no evidence of growth in any vessels inoculated with the product during the repeat test the product passes the test for sterility. This interpretation applies even if growth occurs in negative product control vessels.

498. If there is evidence of growth in the test vessels the product fails the test for sterility. Further testing is not permitted under any circumstances.

499. If two consecutive tests on the same product give evidence of growth in control vessels, or consecutive working sessions give evidence of growth in controls, or there is any other evidence of breakdown in testing methods, then there should be a complete review of all facilities and testing procedures to determine the cause of the contamination. Further tests on samples should be suspended until the review is completed.

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