EP7.0--5.1.3微生物防腐功效测试

EUROPEAN PHARMACOPOEIA 7.0 5.1.3.Efficacy of antimicrobial

preservation

suspensions may be presented in sealed ampoules.Biological indicators are prepared in such a way that they can be stored under defined conditions;an expiry date is set.

Micro-organisms of the same bacterial species as the bacteria used to manufacture the biological indicators may be inoculated directly into a liquid product to be sterilised or into a liquid product similar to that to be sterilised.In this case,it must be demonstrated that the liquid product has no inhibiting effect on the spores used,especially as regards their germination.A biological indicator is characterised by the name of the

species of bacterium used as the reference micro-organism,the number of the strain in the original collection,the number of viable spores per carrier and the D -value.The D -value is the value of a parameter of sterilisation (duration or absorbed dose)required to reduce the number of viable organisms to 10per cent of the original number.It is of significance only under precisely defined experimental conditions.Only the stated micro-organisms are present.Biological indicators consisting of more than one species of bacteria on the same carrier may be https://www.wendangku.net/doc/9a1453475.html,rmation on the culture medium and the incubation conditions is supplied.

It is recommended that the indicator organisms are placed at the locations presumed,or wherever possible,found by previous physical measurement to be least accessible to the sterilising agent.After exposure to the sterilising agent,aseptic technique is used to transfer carriers of spores to the culture media,so that no contamination is present at the time of examination.Biological indicators that include an ampoule of culture medium placed directly in the packaging protecting the inoculated carrier may be used.

A choice of indicator organisms is made such that:

a)the resistance of the test strain is suitable for the particular sterilisation method and is great compared to the resistance of micro-organisms potentially contaminating the product;b)the test strain is non-pathogenic;c)the test strain is easy to culture.

After incubation,growth of the reference micro-organisms subjected to a sterilisation procedure indicates that the procedure has been unsatisfactory.

Steam sterilisation .The use of biological indicators intended for steam sterilisation is recommended for the validation of sterilisation cycles.Spores of Geobacillus stearothermophilus (for example,ATCC 7953,NCTC 10007,NCIMB 8157or CIP 52.81)or other strains of micro-organisms having demonstrated equivalent performance are recommended.The number of viable spores exceeds 5×105per carrier.The D -value at

121°C is not less than 1.5min.It is verified that exposing the biological indicators to steam at 121±1°C for 6min leaves revivable spores,and that there is no growth of the reference micro-organisms after the biological indicators have been exposed to steam at 121±1°C for 15min.

Dry-heat sterilisation .Spores of Bacillus atrophaeus (for example,ATCC 9372,NCIMB 8058or CIP 77.18)or other strains of micro-organisms having demonstrated equivalent performance are recommended for the preparation of biological indicators.The number of viable spores exceeds 1×106per carrier and the D -value at 160°C is not less than 2.5min.Dry heat at temperatures greater than 220°C is frequently used for sterilisation and depyrogenation of glassware.In this case,demonstration of a 3log reduction in heat-resistant bacterial endotoxin can be used as a replacement for biological indicators.Ionising radiation sterilisation .Biological indicators may be used to monitor routine operations,as an additional possibility to assess the effectiveness of the set dose of radiation energy,especially in the case of accelerated electron sterilisation.The spores of Bacillus pumilus (for example,ATCC 27142,NCTC 10327,NCIMB 10692or CIP 77.25)or other strains of micro-organisms having demonstrated equivalent performance are recommended.The number of viable spores exceeds 1×107per carrier.The D -value is not less than 1.9kGy.It is verified that there is no growth of the reference micro-organisms after the biological indicators have been exposed to 25kGy (minimum absorbed dose).

Gas sterilisation .The use of biological indicators is necessary for all gas sterilisation procedures,both for the validation of the cycles and for routine operations.Gas sterilisation is widely used for medical devices,isolators,chambers,https://www.wendangku.net/doc/9a1453475.html,e for such purposes is outside the scope of the European Pharmacopoeia.The use of spores of Bacillus atrophaeus (for example,ATCC 9372,NCIMB 8058or CIP 77.18)or other strains of micro-organisms having demonstrated equivalent performance is recommended for ethylene oxide.The number of viable spores exceeds 1×106per carrier.The parameters of resistance are the following:the D -value is not less than 2.5min for a test cycle involving 600mg/L of ethylene oxide,at 54°C and at 60per cent relative humidity.It is verified that there is no growth of the reference micro-organisms after the biological indicators have been exposed to the test cycle described above for 25min and that exposing the indicators to a reduced temperature cycle (600mg/L,30°C and 60per cent relative humidity)for 50min leaves revivable spores.

01/2011:50103

5.1.3.EFFICACY OF ANTIMICROBIAL PRESERVATION If a pharmaceutical

antimicrobial activity,antimicrobial preservatives may be added,particularly to aqueous preparations,to prevent proliferation or to limit microbial contamination which,during normal conditions of storage and use,particularly for multidose containers,could occur in a product and present a hazard to the patient from infection and spoilage of the preparation.

Antimicrobial preservatives must not be used as a substitute for good manufacturing practice.

The efficacy of an antimicrobial preservative may be enhanced or diminished by the active constituent of the preparation or by the formulation in which it is incorporated or by the container and closure used.The antimicrobial activity of the preparation in its final container is investigated over the period of validity to ensure that such activity has not been impaired by storage.Such investigations may be carried out on samples removed from the final container immediately prior to testing.

During development of a pharmaceutical preparation,it shall be demonstrated that the antimicrobial activity of the preparation as such or,if necessary,with the addition of a suitable

preservative or preservatives provides adequate protection from adverse effects that may arise from microbial contamination or proliferation during storage and use of the preparation.

The efficacy of the antimicrobial activity may be demonstrated by the test described below.The test is not intended to be used for routine control purposes.

TEST FOR EFFICACY OF ANTIMICROBIAL PRESERVATION The test consists of challenging the preparation,wherever possible in its final container,with a prescribed inoculum of suitable micro-organisms,storing the inoculated preparation at a prescribed temperature,withdrawing samples from the container at specified intervals of time and counting the organisms in the samples so removed.

The preservative properties of the preparation are adequate if,in the conditions of the test,there is a significant fall or no increase,as appropriate,in the number of micro-organisms in the inoculated preparation after the times and at the

temperatures prescribed.The acceptance criteria,in terms of decrease in the number of micro-organisms with time,vary for different types of preparations according to the degree of protection intended (see Tables 5.1.3.-1/2/3).

General Notices (1)apply to all monographs and other texts

505

5.1.3.Efficacy of antimicrobial preservation EUROPEAN PHARMACOPOEIA

7.0

Test micro-organisms

Pseudomonas aeruginosa ATCC9027;NCIMB8626;CIP82.118. Staphylococcus aureus ATCC6538;NCTC10788;

NCIMB9518;CIP4.83.

Candida albicans ATCC10231;NCPF3179;IP48.72. Aspergillus brasiliensis ATCC16404;IMI149007;IP1431.83. Single-strain challenges are used and the designated

micro-organisms are supplemented,where appropriate,by other strains or species that may represent likely contaminants to the preparation.It is recommended,for example,that Escherichia coli(ATCC8739;NCIMB8545;CIP53.126)is used for all oral preparations and Zygosaccharomyces rouxii (NCYC381;IP2021.92)for oral preparations containing a high concentration of sugar.

Preparation of inoculum

Preparatory to the test,inoculate the surface of casein soya bean digest agar(2.6.12)for bacteria or Sabouraud-dextrose agar without the addition of antibiotics(2.6.12)for fungi,

with the recently grown stock culture of each of the specified micro-organisms.Incubate the bacterial cultures at30-35°C for 18-24h,the culture of C.albicans at20-25°C for48h,and the culture of A.brasiliensis at20-25°C for1week or until good sporulation is obtained.Subcultures may be needed after revival before the micro-organism is in its optimal state,but it is recommended that their number be kept to a minimum.

To harvest the bacterial and C.albicans cultures,use a sterile suspending fluid,containing9g/L of sodium chloride R,for dispersal and transfer of the surface growth into a suitable vessel.Add sufficient suspending fluid to reduce the microbial count to about108micro-organisms per millilitre.To harvest the A.brasiliensis culture,use a sterile suspending fluid containing 9g/L of sodium chloride R and0.5g/L of polysorbate80R and adjust the spore count to about108per millilitre by adding the same solution.

Remove immediately a suitable sample from each suspension and determine the number of colony-forming units per millilitre in each suspension by plate count or membrane filtration (2.6.12).This value serves to determine the inoculum and

the baseline to use in the test.The suspensions shall be used immediately.

METHOD

To count the viable micro-organisms in the inoculated products, use the agar medium used for the initial cultivation of the respective micro-organisms.

Inoculate a series of containers of the product to be examined, each with a suspension of one of the test organisms to give an inoculum of105to106micro-organisms per millilitre or per gram of the preparation.The volume of the suspension of inoculum does not exceed1per cent of the volume of the product.Mix thoroughly to ensure homogeneous distribution.

Maintain the inoculated product at20-25°C,protected from light.Remove a suitable sample from each container,typically 1mL or1g,at zero hour and at appropriate intervals according to the type of the product and determine the number of viable micro-organisms by plate count or membrane filtration(2.6.12). Ensure that any residual antimicrobial activity of the product is eliminated by dilution,by filtration or by the use of a specific inactivator.When dilution procedures are used,due allowance is made for the reduced sensitivity in the recovery of small numbers of viable micro-organisms.When a specific inactivator is used,the ability of the system to support the growth of the test organisms is confirmed by the use of appropriate controls. The procedure is validated to verify its ability to demonstrate the required reduction in count of viable micro-organisms. ACCEPTANCE CRITERIA

The criteria for evaluation of antimicrobial activity are given in Tables5.1.3.-1/2/3in terms of the log reduction in the number of viable micro-organisms against the value obtained for the inoculum.

Table5.1.3.-1.-Parenteral preparations,eye preparations, intrauterine preparations and intramammary preparations

Log reduction

6h24h7d14d28d

A23--NR Bacteria

B-13-NI

A--2-NI Fungi

B---1NI NR:no recovery.

NI:no increase in number of viable micro-organisms compared to the previous reading.

The A criteria express the recommended efficacy to be achieved. In justified cases where the A criteria cannot be attained,for example for reasons of an increased risk of adverse reactions, the B criteria must be satisfied.

Table5.1.3.-2.-Ear preparations,nasal preparations, preparations for cutaneous application and preparations for

inhalation

Log reduction

2d7d14d28d

A23-NI Bacteria

B--3NI

A--2NI Fungi

B--1NI NI:no increase in number of viable micro-organisms compared to the previous reading.

The A criteria express the recommended efficacy to be achieved. In justified cases where the A criteria cannot be attained,for example for reasons of an increased risk of adverse reactions, the B criteria must be satisfied.

Table5.1.3.-3.-Oral preparations,oromucosal preparations

and rectal preparations

Log reduction

14d28d

Bacteria3NI

Fungi1NI

NI:no increase in number of viable micro-organisms compared to the previous reading.

The above criteria express the recommended efficacy to be achieved.

506See the information section on general monographs(cover pages)