Sterility testing repeat

The British Pharmacopoeia makes an allowance for acddenlal contamination which may arise during the execution of a sterility test by aUowing the test to be repeated. Under these circumstances the following rules apply. 

Perform a pressure hold test with the nonsterilized filter. Expose the filter to the chosen sterilization conditions. Repeat the pressure hold test with die sterilized filter. For pressure hold test conditions, follow the recommendations of the filter supplier. 

Under certain circumstances a sterility test may be repeated, but the only justification for repeating the test is unequivocal evidence that the first test was... 

Running records should be maintained of sterility test results. The frequency of any repeat testing can give an indication of the confidence to be placed in the testing procedure. 

For the Rideal-Walker test, prepare four suitable serial dilutions of the disinfectant and one of the standard phenol in 5-ml amounts in 5 X f in. sterile test-tubes, and cool in a water-bath to 17-18°. At halfminute intervals, add 0-2 ml of the twenty-four-hour culture of Salm. typhi (also cooled to 17 18°) in turn to each of the tubes, and then at subsequent two and a half, five, seven and a half, and ten minute intervals remove one standard loopful of the mixture to a 5-ml tube of the medium, incubate at 37 for forty-eight to seventy-two hours and record the growths obtained. It may be necessary to repeat the test with another 5 dilutions, but using a different phenol dilution, in order to obtain a satisfactory end-point. Calculate the Rideal-Walker Coefficient by dividing the numerical value of the dilution of the disinfectant which... 

The interpretation of sterility results is divided into two stages by the USP relative to the type of sterility failure if one occurs. If sterility failure of the test samples occurred because of improper aseptic technique or as a fault of the test itself, stage 1 may be repeated with the same sample size. Sample size is doubled in a stage 2 testing, which is performed if microbial growth is observed in stage 1 and there is no reason to believe that the test was invalid. The only absolute method to guarantee the sterility of a batch would be to test every vial or ampoule. 

Repeat this for each test organism. For positive control use sterile DW instead of sanitizer solution. For negative control use sterile strips. Swab each strip thoroughly with a premoistened Ca alginate swab and place in Lecithin broth (or other suitable neutralizer). Vortex for 30 sec, then perform the usual pour plate method. 

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. 

For sterilization validation, the sterilization process must be defined and the parameters described in writing. The limits and test procedures must be described. The process is then run and the records cross-checked against the parameters. The results are then evaluated. This process is repeated three times to prove the reproducibility of the process. 

The avoidance of false-negatives is addressed at length in the pharmacopeias. Each batch of medium used in the Test for Sterility must have been shown to be capable of supporting the growth of low inocula of a specified array of microorganisms. Each Test for Sterility applicable to each specific product must be validated by repeated demonstration that the viabilities of low inocula of a specified array of microorganisms are not inhibited by product traces contained in the medium (direct inoculation) or on the membrane filter. 

All the controls may be conducted either before, or in parallel with, the test itself, providing that the same batches of media are used for both. If the controls are carried out in parallel with the tests and one of the controls gives an unexpected result, the test for sterility may be declared invalid, and, when the problem is resolved, the test may be repeated. 

Striga seeds were initially surface sterilized with 1% aqueous NaOCl, followed by two deionized water rinses and then were pre-incubated in lOmL of deionized water in the dark at 28°C for 10 days. Samples of pre-incubated seeds were collected on 5 /hn Metricel filters (Gelman Type GA-1) and floated in lOmL test solution or control solution (either 0.1% dimethyl sulfoxide or deionized water). For the germination incubations, seeds and test solution were transferred to 96 well plastic culture dishes (0.4 mL/well and 4 replicates of 8 wells each for a given assay). The Striga seeds were then incubated in the dark for 3 days at 28°C before evaluation of germination (radicle protrusion) under 40X magnification. Each experiment was repeated at least twice. Where necessary, test solution pH was adjusted to 6.8 with 0.IN KOH. 

A sterile product incorporated a preservative system to cover withdrawal of a multidose liquid product. The product passed the USP XX microbial challenge test when first made and packed. After six months it was noticed that the level of ethylenediamine tetra acetate (EDTA) had significantly reduced, possibly by chelation with heavy metals + surface adsorption onto the plastic. A repeat microbial challenge test was not passed. It was theorised that this was due to slight preservative loss coupled to the loss of EDTA which tended to enhance the preservative efficacy, i.e. a chemical + physical change (preservative adsorption) had created a drop in preservative efficacy. Changing the EDTA/preservative levels was subsequently necessary in order to meet the microbial challenge test over the full shelf-life period. 

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