hwýUROPEAN
`PHARMACOPOEIA
`SEVENTH EDITION
`
`Volume 1
`
`Published in accordance with the
`Convention on the Elaboration of a European Pharmacopoeia
`European Treaty Series No. 50
`
`ý
`
`EXHIBIT
`BLS
`
`Deponent
`
`Date f
`/WWW
`DEPO ooKp ýý
`
`European Directorate
`HealthCare
`Quality of Medicines
`
`for the
`
`Council of Europe
`
`Strasbourg
`
`InnoPharma EX1060
`IPR2015-00903
`
`IPR2015-00902
`
`LUPIN068837
`
`

`
`Pharmacopoeia
`The European
`is published
`HealthCare of the Council of Europe EDQM.
`
`by the Directorate
`
`for
`
`the Quality of Medicines
`
`Council of Europe 67075 Strasbourg Cedex France - 2010
`
`All rights reserved. Apart from any fair dealing for the purposes of research or private study this
`publication may not be reproduced stored or transmitted in any form or by any means without
`the
`prior permission in writing of the publisher.
`
`ISBN 978-92-871-6700-2
`
`LUPIN068838
`
`Page 2
`
`

`
`EUROPEAN PHARMACOPOEIA
`
`7.0
`
`2.6.12. Microbial enumeration tests
`
`the animal
`
`the rectal
`
`Introduce
`
`limits.
`
`it
`
`2. GENERAL PROCEDURES
`
`Carry out the determination under conditions designed to
`avoid extrinsic microbial contamination of the product to be
`examined. The precautions
`taken to avoid contamination must
`be such that they do not affect any micro-organisms that are to
`be revealed in the test.
`
`If surface-active
`
`it
`
`3. ENUMERATION
`
`METHODS
`
`from loss of body heat and maintain it so that
`temperature remains within physiological
`a cannula into the trachea.
`Insert a cannula filled
`9 g/L solution of sodium chloride into the
`with a heparinised
`to a device capable of
`common carotid artery and connect
`giving a continuous record of the blood pressure.
`Insert into the
`femoral vein another cannula filled with a heparinised 9 g/L
`the product to be examined
`has antimicrobial activity this is
`If
`solution of sodium chloride through which can be injected the
`insofar as possible removed or neutralised.
`If inactivators are
`to be examined.
`solutions of histamine and of the substance
`used for this purpose their efficacy and their absence of toxicity
`Determine the sensitivity of the animal to histamine by injecting
`intravenously at regular intervals doses of histamine solution R for micro-organisms must be demonstrated.
`substances are used for sample preparation
`corresponding to 0.1 dig and 0.15 pg of histamine base per
`the lower dose at least 3 times.
`kilogram of body mass. Repeat
`their absence of toxicity for micro-organisms and their
`compatibility with inactivators used must be demonstrated.
`injections not less than
`Administer the second and subsequent
`1 min after the blood pressure has returned to the level
`was at immediately before the previous injection. The animal
`is used for the test only if a readily discernible decrease in
`for the lower dose is obtained
`blood pressure that is constant
`the higher dose causes greater responses. Dissolve the
`and if
`in sufficient of a 9 g/L solution
`substance to be examined
`to give the
`of sodium chloride or other prescribed
`solvent
`concentration.
`
`prescribed
`
`Inject intravenously per kilogram
`
`of body mass 1.0 mL of histamine solution R followed
`by
`2 successive injections of the prescribed amount of the solution
`1.0 mL of histamine solution R.
`to be examined and finally
`The second third and fourth injections are given not less than
`1 min after the blood pressure has returned to the level
`it was at
`immediately before the preceding
`injection. Repeat
`this series
`of injections twice and conclude the test by giving 1.5 mL of
`histamine solution R per kilogram of body mass.
`the response to 1.5 mL of histamine solution R per kilogram
`of body mass is not greater than that to 1.0 mL the test is
`invalid. The substance to be examined
`the mean
`fails the test if
`
`If
`
`of the series of responses to the substance is greater than the
`mean of the responses to 1.0 mL of histamine solution R per
`kilogram of body mass or if any one dose of the substance
`dose
`causes a greater depressor
`response than the concluding
`of the histamine solution. The test animal must not be used in
`substances
`the second criterion
`another test for depressor
`the response to the high dose of histamine given
`applies or if
`after the administration of the substance to be examined is
`less than the mean response to the low doses of histamine
`previously injected.
`
`if
`
`Use the membrane filtration method or the plate-count methods
`The most-probable-number MPN method is
`as prescribed.
`generally the least accurate method for microbial counts
`however
`for certain product groups with a very low bioburden
`it may be the most appropriate method.
`The choice of method is based on factors such as the nature
`of the product and the required limit of micro-organisms. The
`chosen method must allow testing of a sufficient sample size to
`The suitability of the
`judge compliance with the specification.
`method chosen must be established.
`
`TEST SUITABILITY OF THE
`4. GROWTH PROMOTION
`COUNTING METHOD AND NEGATIVE CONTROLS
`4-1. GENERAL CONSIDERATIONS
`
`The ability of the test to detect micro-organisms in the presence
`to be tested must be established.
`of product
`
`Suitability must be confirmed if a change in testing performance
`the outcome of the test
`or the product which may affect
`introduced.
`
`is
`
`4-2. PREPARATION OF TEST STRAINS
`Use standardised
`stable suspensions of test strains or prepare
`them as stated below. Seed lot culture maintenance
`techniques
`seed-lot systems are used so that the viable micro-organisms
`used for inoculation are not more than 5 passages removed
`from the original master seed-lot. Grow each of the bacterial
`as described in Table 2.6.121.
`and fungal test strains separately
`
`solution pH 7.0 or
`Use buffered sodium chloride-peptone
`phosphate buffer solution pH 7.2 to make test suspensions to
`80
`suspend A. brasiliensis spores 0.05 per cent of polysorbate
`may be added to the buffer. Use the suspensions within
`2 h or within 24 h if stored at 2-8 C. As an alternative to
`2.6.12. MICROBIOLOGICAL
`preparing and then diluting a fresh suspension of vegetative
`EXAMINATION OF NON-STERILE
`cells of A. brasiliensis or B. subtilis a stable spore suspension
`an
`of
`PRODUCTS MICROBIAL ENUMERATION suspension
`iat
`The stable spore
`iss used forr t
`suspension may be maintained at 2-8 C for a validated
`TESTS1
`period
`
`is
`
`t
`
`inoculation.
`
`07/201020612
`
`1.
`
`INTRODUCTION
`
`The tests described hereafter will allow quantitative enumeration
`of mesophilic bacteria and fungi that may grow under aerobic
`conditions.
`
`The tests are designed primarily to determine whether
`a substance or preparation complies with an established
`for microbiological quality. When used for such
`specification
`follow the instructions given below including the
`purposes
`number of samples to be taken and interpret
`the results as
`stated below.
`
`The methods
`
`are not applicable
`
`to products containing viable
`
`micro-organisms as active ingredients.
`
`Alternative microbiological procedures including automated
`methods may be used provided
`to the
`that their equivalence
`Pharmacopoeia method has been demonstrated.
`
`of time.
`
`4-3. NEGATIVE CONTROL
`To verify testing conditions a negative control
`is performed
`using the chosen diluent
`in place of the test preparation. There
`must be no growth of micro-organisms. A negative control
`is also performed when testing the products
`as described
`in
`section 5. A failed negative control requires an investigation.
`4-4. GROWTH PROMOTION OF THE MEDIA
`Test each batch of ready-prepared medium and each batch of
`medium prepared either from dehydrated medium or from the
`ingredients described.
`
`Inoculate portions/plates of casein soya bean digest broth and
`casein soya bean digest agar with a small number not more than
`100 CFU of the micro-organisms indicated
`in Table 2.6.121
`using a separate portion/plate of medium for each.
`Inoculate
`agar with a small number not
`plates of Sabouraud-dextrose
`
`1 This chapter has undergone
`
`pharmacopoeial
`
`harmonisation.
`
`See chapter
`
`5.8. Pharmacopoeial
`
`harmonisation.
`
`General Notices 1 apply to all monographs and other texts
`
`-
`
`163
`
`LUPIN068839
`
`Page 3
`
`

`
`2.6.12. Microbial enumeration tests
`
`EUROPEAN PHARMACOPOEIA
`
`7.0
`
`Micro-organism
`
`Preparation
`
`of test
`
`Growth promotion
`
`strain
`
`Suitability of counting method in the
`presence of the product
`
`Table 2.6.12.-1. - Preparation and use of test micro-organisms
`
`Staphylococcus
`
`aureus
`
`such as
`
`ATCC 6538
`
`NCIMB 9518
`
`CIP 4.83
`NBRC 13276
`Pseudomonas
`
`aeruginosa
`
`such as
`
`ATCC 9027
`
`NCIMB 8626
`
`CIP 82.118
`NBRC 13275
`
`Bacillus subtilis
`such as
`
`ATCC 6633
`
`NCIMB 8054
`
`Casein soya bean digest
`agar or casein soya
`bean digest broth
`
`30-35 C
`
`18-24 h
`
`Casein soya bean digest
`agar or casein soya
`bean digest broth
`
`30-35 C
`
`18-24h
`
`Casein soya bean digest
`agar or casein soya
`bean digest broth
`
`30-35 C
`
`18-24 h
`
`Total aerobic microbial
`
`count
`
`bean digest
`Casein soya
`agar and casein soya
`bean digest broth
`
`s 100 CFU
`
`3035 C
`
`_ 3 days
`
`Casein
`
`bean digest
`soya
`agar and casein soya
`bean digest broth
`
`5 100 CFU
`
`30-35 C
`
`3 days
`
`Casein soya bean digest
`agar and casein soya
`bean digest broth
`
`_ 100 CFU
`
`30-35 C
`
`C 3 days
`
`Total yeasts and
`moulds count
`
`Total aerobic microbial
`
`count
`
`Total yeasts and
`moulds count
`
`-
`
`Casein soya bean digest
`agar/MPN casein soya
`bean digest broth
`100 CFU
`30-35 C
`
`_ 3 days
`
`Casein soya bean digest
`agar/MPN casein soya
`bean digest broth
`
`5 100 CFU
`
`30-35 C
`
`3 days
`
`Casein soya bean digest
`agar/MPN casein soya
`bean digest broth
`
`- 100 CFU
`
`30-35 C
`
`a 3 days
`
`Sabouraud-dextrose
`
`bean
`
`Sabouraud-dextrose
`
`CIP 52.62
`NBRC 3134
`Candida albicans
`
`such as
`
`ATCC 10231
`
`NCPF 3179
`
`IP 48.72
`NBRC 1594
`
`Sabouraud-dextrose
`
`agar or Sabouraud-
`dextrose broth 20-25 C
`2-3 days
`
`Casein soya bean
`digest agar
`
`_ 100 CFU
`
`30-35 C
`
`5 days
`
`agar
`
`_ 100 CFU
`20-25 C
`s 5 days
`
`Casein soya
`digest agar
`
`_ 100 CFU
`30-35 C
`5 5 days
`MPN not applicable
`
`agar
`
`s 100 CFU
`20-25 C
`
`s 5 days
`
`Aspergillus brasiliensis
`
`Sabouraud-dextrose
`
`Casein soya bean
`
`Sabouraud-dextrose
`
`Casein soya bean
`
`Sabouraud-dextrose
`
`such as
`
`ATCC 16404
`
`IMI 149007
`
`IP 1431.83
`NBRC 9455
`
`agar or potato-dextrose
`
`agar
`
`20-25 C
`
`5-7 days or until good
`sporulation is achieved
`
`digest agar
`
`a 100 CFU
`
`30-35 C
`
`s 5 days
`
`agar
`
`100 CFU
`20-25 C
`5 days
`
`digest agar
`
`s 100 CFU
`30-35 C
`- 5 days
`MPN not applicable
`
`agar
`
`a 100 CFU
`20-25 C
`5 days
`
`more than 100 CFU of the micro-organisms indicated
`2.6.12.-1 using a separate plate of medium for each.
`in the conditions described
`in Table 2.6.121.
`
`in Table
`
`Incubate
`
`For solid media growth obtained must not differ by a factor
`than 2 from the calculated
`value for a standardised
`greater
`inoculum growth of the
`inoculum. For a freshly prepared
`micro-organisms comparable to that previously obtained with
`batch of medium occurs.
`a previously tested and approved
`Liquid media are suitable if clearly visible growth of the
`micro-organisms comparable to that previously obtained with a
`previously tested and approved batch of medium occurs.
`4-5. SUITABILITY OF THE COUNTING METHOD IN THE
`PRESENCE OF PRODUCT
`4-5-1. Preparation of the sample. The method for sample
`preparation depends upon the physical
`of the
`If none of the procedures
`product to be tested.
`can be demonstrated
`to be satisfactory an alternative procedure
`must be developed.
`
`described below
`
`characteristics
`
`Water-soluble
`products. Dissolve or dilute usually a 1 in 10
`in buffered
`dilution is prepared the product to be examined
`solution pH 7.0 phosphate buffer
`sodium chloride-peptone
`solution pH 7.2 or casein soya bean digest broth.
`If necessary
`adjust to pH 6-8. Further dilutions where necessary
`prepared with the same diluent.
`
`are
`
`non-inhibitory sterile surface-active agent heated if necessary to
`not more than 40 C or in exceptional
`cases to not more than
`45 C. Mix carefully and if necessary maintain the temperature
`Add sufficient of the pre-warmed
`in a water-bath.
`chosen
`diluent to make a 1 in 10 dilution of the original product. Mix
`carefully whilst maintaining the temperature for the shortest
`time necessary for the formation of an emulsion. Further serial
`tenfold dilutions may be prepared using the chosen diluent
`containing a suitable concentration
`of sterile polysorbate 80 or
`another non-inhibitory sterile surface-active agent.
`
`Fluids or solids in aerosol form. Aseptically transfer the
`into a membrane filter apparatus or a sterile container
`product
`for further sampling. Use either the total contents or a defined
`number of metered doses from each of the containers
`
`tested.
`
`Transdermal patches. Remove the protective
`cover sheets
`release liners of the transdermal patches and place them
`adhesive side upwards on sterile glass or plastic trays. Cover
`the adhesive surface with a sterile porous material
`for example
`sterile gauze to prevent
`the patches from sticking together and
`transfer the patches to a suitable volume of the chosen diluent
`containing inactivators such as polysorbate
`80 and/or
`Shake the preparation vigorously for at least 30 min.
`
`lecithin.
`
`4-5-2. Inoculation and dilution. Add to the sample prepared as
`described above 4-5-1 and to a control with no test material
`included a sufficient volume of the microbial suspension
`to
`obtain an inoculum of not more than 100 CFU. The volume of
`the suspension of the inoculum should not exceed 1 per cent of
`the volume of diluted product.
`
`Non-fatty products insoluble in water. Suspend the product
`to be examined usually a 1 in 10 dilution is prepared in
`solution pH 7.0 phosphate
`buffered sodium chloride-peptone
`buffer solution pH 7.2 or casein soya bean digest broth. A
`80 may be
`agent such as 1 g/L of polysorbate
`added to assist
`the suspension of poorly wettable
`substances.
`necessary adjust to pH 6-8. Further dilutions where necessary
`are prepared with the same diluent.
`
`surface-active
`
`acceptable microbial recovery from the product
`the lowest possible dilution factor of the prepared sample
`must be used for the test. Where this is not possible due to
`antimicrobial activity or poor solubility further appropriate
`Fatty products. Dissolve in isopropyl myristate sterilised by
`filtration or mix the product to be examined with the minimum protocols must be developed.
`inhibition of growth by the
`If
`sample cannot otherwise be avoided
`necessary quantity of sterile polysorbate 80 or another
`the aliquot of the microbial
`
`If
`
`To demonstrate
`
`164
`
`See the information section
`
`on general monographs cover pages
`
`LUPIN068840
`
`Page 4
`
`

`
`EUROPEAN PHARMACOPOEIA
`
`7.0
`
`2.6.12. Microbial enumeration tests
`
`suspension may be added after neutralisation dilution or
`
`filtration.
`
`of antimicrobial activity. The
`4-5-3. Neutralisation/removal
`number of micro-organisms recovered
`from the prepared
`in 4-5-2 and incubated
`sample diluted as described
`following
`to the number of
`in 4-5-4 is compared
`described
`the procedure
`micro-organisms recovered from the control preparation.
`If growth is inhibited reduction by a factor greater than 2 then
`modify the procedure
`for the particular enumeration test
`to
`ensure the validity of the results. Modification of the procedure
`
`may include for example 1 an increase in the volume of
`the diluent or culture medium 2 incorporation of specific
`or general neutralising agents into the diluent 3 membrane
`filtration or 4 a combination of the above measures.
`
`Neutralising agents. Neutralising agents may be used to
`neutralise the activity of antimicrobial agents Table 2.6.122.
`They may be added to the chosen diluent or the medium
`If used their efficacy and their
`preferably before sterilisation.
`absence of toxicity for micro-organisms must be demonstrated
`by carrying out a blank with neutraliser and without product.
`
`Table 2.6.12.-2.
`
`- Common neutralising agents for interfering
`substances
`
`Interfering substance
`
`Clutaraldehyde
`
`mercurials
`
`Potential neutralising
`method
`
`Sodium hydrogensulfite
`sodium bisulfite
`
`Phenolics alcohol aldehydes
`
`sorbate
`
`Dilution
`
`Aldehydes
`
`Quaternary Ammonium Compounds
`QACs parahydroxybenzoates
`parabens
`bis-biguanides
`
`QACs iodine parabens
`
`Mercurials
`
`Glycine
`
`Lecithin
`
`Polysorbate
`
`Thioglycollate
`
`Mercurials
`
`halogens aldehydes
`
`Thiosulfate
`
`EDTA edetate
`
`Mg2 or Cal
`
`ions
`
`If no suitable neutralising method can be found it can be
`assumed that the failure to isolate the inoculated organism is
`attributable to the microbicidal activity of the product. This
`information serves to indicate that the product is not likely to
`be contaminated with the given species of the micro-organism.
`However
`is possible that the product only inhibits some
`of the micro-organisms specified herein but does not inhibit
`others not included amongst the test strains or for which the
`Then perform the test with the
`latter are not representative.
`highest dilution factor compatible with microbial growth and
`the specific acceptance criterion.
`
`it
`
`4-5-4-1. Membrane filtration. Use membrane filters having a
`nominal pore size not greater than 0.45 pm. The type of filter
`material is chosen such that
`the bacteria-retaining efficiency
`not affected by the components of the sample to be investigated.
`For each of the micro-organisms listed one membrane filter
`is used.
`
`is
`
`Transfer a suitable amount of the sample prepared
`as described
`under 4-5-1 to 4-5-3 preferably representing 1 g of the product
`large numbers of CFU are expected to the membrane
`or less if
`immediately and rinse the membrane filter with an
`filter
`filter
`appropriate volume of diluent.
`For the determination of total aerobic microbial count TAMC
`transfer the membrane filter
`to the surface of casein soya
`bean digest agar. For the determination of total combined
`
`General Notices 1 apply to all monographs and other texts
`
`reading of the results is difficult or uncertain owing to the
`nature of the product to be examined subculture in the same
`broth or in casein soya bean digest agar for 1-2 days at the
`same temperature and use these results. Determine the most
`probable number of micro-organisms per gram or millilitre
`the product to be examined
`from Table 2.6.123.
`4-6. RESULTS AND INTERPRETATION
`4-5-4. Recovery of micro-organism in the presence of product. When verifying the suitability of. the membrane
`filtration
`For each of the micro-organisms listed separate tests are
`method or the plate-count method a mean count of any of the
`performed. Only micro-organisms of the added test strain are
`test organisms not differing by a factor greater than 2 from
`counted.
`the value of the control defined in 4-5-2 in the absence of the
`product must be obtained. When verifying the suitability of the
`MPN method the calculated
`value from the inoculum must be
`within 95 per cent confidence
`with the control.
`
`yeasts/moulds count TYMC transfer the membrane to the
`the plates as
`agar.
`in Table 2.6.12.-1. Perform the counting.
`
`surface of Sabouraud-dextrose
`
`Incubate
`
`indicated
`
`4-5-4-2. Plate-count methods. Perform plate-count methods at
`for each medium and use the mean count of
`
`least in duplicate
`the result.
`
`4-5-4-2-1. Pour-plate method
`For Petri dishes 9 cm in diameter add to the dish 1 mL of the
`sample prepared as described under 4-5-1 to 4-5-3 and 15-20 mL
`of casein soya bean digest agar or Sabouraud-dextrose
`agar
`both media being at not more than 45 C. If
`larger Petri dishes
`are used the amount of agar medium is increased accordingly.
`For each of the micro-organisms listed in Table 2.6.121 at
`least 2 Petri dishes are used.
`Incubate
`the plates as indicated
`in Table 2.6.121. Take the arithmetic mean of the counts
`the number of CFU in the original
`per medium and calculate
`inoculum.
`
`4-5-4-2-2. Surface-spread method
`For Petri dishes 9 cm in diameter add 15-20 mL of casein soya
`agar at about 45 C to
`bean digest agar or Sabouraud-dextrose
`If larger Petri dishes are
`each Petri dish and allow to solidify.
`used the volume of the agar is increased accordingly. Dry the
`plates for example in a laminar-air-flow cabinet or an incubator.
`For each of the micro-organisms listed in Table 2.6.12.-1 at least
`2 Petri dishes are used. Spread a measured volume of not
`than 0.1 mL of the sample prepared as described under 4-5-1
`the surface of the medium. Incubate
`and count
`to 4-5-3 over
`as prescribed under 4-5-4-2-1.
`4-5-4-3. Most-probable-number MPN method. The precision
`and accuracy of the MPN method is less than that of the
`membrane
`filtration method or the plate-count method.
`Unreliable results are obtained particularly for the enumeration
`of moulds. For these reasons the MPN method is reserved for
`the enumeration of TAMC in situations where no other method
`the use of the method is justified proceed as
`is available.
`
`less
`
`If
`
`follows.
`
`Prepare a series of at least 3 serial tenfold dilutions of the
`product as described under 4-5-1 to 4-5-3. From each level of
`dilution 3 aliquots of 1 g or 1 mL are used to inoculate
`3 tubes
`with 9-10 mL of casein soya bean digest broth.
`If necessary
`agent such as polysorbate 80 or an inactivator
`surface-active
`antimicrobial agents may be added to the medium. Thus if 3
`levels of dilution are prepared 9 tubes are inoculated.
`
`a
`
`of
`
`Incubate
`
`all
`
`tubes at 30-35 C for not more than 3 days.
`
`If
`
`of
`
`limits of the results obtained
`
`be met for one or more of the
`If the above criteria cannot
`organisms tested with any of the described methods the method
`and test conditions that come closest
`to the criteria are used to
`test the product.
`
`5. TESTING OF PRODUCTS
`5-1. AMOUNT USED FOR THE TEST
`Unless otherwise prescribed use 10 g or 10 mL of the product
`referred to above.
`to be examined taken with the precautions
`form sample 10 containers. For
`For fluids or solids in aerosol
`sample 10 patches.
`transdermal patches
`
`165
`
`LUPIN068841
`
`Page 5
`
`

`
`2.6.12. Microbial enumeration tests
`
`EUROPEAN PHARMACOPOEIA
`
`7.0
`
`Table 2.6.12.-3.
`
`- Most-probable-number values of
`
`micro-organisms
`
`Observed combinations
`
`of numbers of
`
`tubes showing growth in each set
`Number of grams or millilitres
`product per tube
`
`of
`
`0.1
`
`0.01
`
`0.001
`
`MPN per
`gram or per
`of
`
`millilitre
`
`product
`
`95 per cent
`e
`confidence
`mats
`
`0
`
`0
`
`0
`
`0
`
`0
`
`0
`
`1
`
`1
`
`0
`
`0
`
`1
`
`1
`
`2
`
`3
`
`0
`
`0
`
`0
`
`1
`
`0
`
`1
`
`0
`
`0
`
`0
`
`1
`
`3
`
`3
`
`3
`
`6.1
`
`6.2
`
`9.4
`
`3.6
`
`7.2
`
`0-9.4
`
`0.1-9.5
`
`0.1-10
`
`1.2-17
`
`1.2-17
`
`3.5-35
`
`0.2-17
`
`1.2-17
`
`preparations not presented in dose units is less than 1 mg.
`In
`these cases the amount to be tested is not less than the amount
`in 10 dosage units or 10 g or 10 mL of the product.
`present
`For materials used as active substances where sample quantity
`less than 1000 mL
`is limited or batch size is extremely small i.e.
`or 1000 g the amount tested shall be 1 per cent of the batch
`unless a lesser amount is prescribed or justified and authorised.
`For products where the total number of entities in a batch is less
`than 200 e.g. samples used in clinical trials the sample size
`may be reduced to 2 units or I unit if
`the size is less than 100.
`the samples at random from the bulk material or from
`Select
`the available containers
`of the preparation.
`To obtain the
`required quantity mix the contents of a sufficient number of
`to provide the sample.
`containers
`5-2. EXAMINATION OF THE PRODUCT
`
`5-2-1. Membrane filtration
`
`1
`
`1
`
`1
`
`1
`
`1
`
`1
`
`2
`
`2
`
`2
`
`2
`
`0
`
`1
`
`1
`
`2
`
`2
`
`3
`
`0
`
`0
`
`0
`
`1
`
`1
`
`2
`
`0
`
`1
`
`0
`
`1
`
`0
`
`0
`
`1
`
`2
`
`0
`
`1
`
`11
`
`7.4
`
`11
`
`11
`
`15
`
`16.
`
`9.2
`
`14
`
`20
`
`15
`
`20
`
`4-35
`
`1.3-20
`
`4-35
`
`4-35
`
`5-38
`
`5-38
`
`1.5-35
`
`4-35
`
`5-38
`
`4-38
`
`5-38
`
`agar.
`
`designed to allow the transfer of the
`Use a filtration apparatus
`to the medium. Prepare the sample using a method that
`filter
`has been shown suitable as described in section 4 and transfer
`the appropriate amount to each of 2 membrane filters and filter
`shown
`immediately. Wash each filter
`following the procedure
`to be suitable.
`For the determination of TAMC transfer one of the membrane
`filters to the surface of casein soya bean digest agar. For the
`determination of TYMC transfer the other membrane
`to the
`surface of Sabouraud-dextrose
`Incubate the plate of casein
`soya bean digest agar at 30-35 C for 3-5 days and-the plate of
`agar at 20-25 C for 5-7 days. Calculate the
`Sabouraud-dextrose
`number of CFU per gram or per millilitre
`When examining transdermal patches
`10 per cent of the
`volume of the preparation described under 4-5-1 separately
`through each of 2 sterile filter membranes. Transfer one
`membrane to casein soya bean digest agar for TAMC and the
`other membrane to Sabouraud-dextrose
`agar for TYMC.
`
`of product.
`
`filter
`
`2
`
`2
`
`2
`
`2
`
`2
`
`2
`
`2
`
`3
`
`3
`
`3
`
`3
`
`3
`
`1
`
`2
`
`2
`
`2
`
`3
`
`3
`
`0
`
`0
`
`0
`
`1
`
`1
`
`1
`
`2
`
`0
`
`1
`
`2
`
`0
`
`1
`
`o
`
`1
`
`2
`
`0
`
`1
`
`27
`
`21
`
`28
`
`35
`
`29
`
`36
`
`23
`
`38
`
`64
`
`43
`
`75
`
`9-94
`
`5-40
`
`9-94
`
`994
`
`9.94
`
`9 94
`
`5-94
`
`9-104
`
`16.181
`
`9-181
`
`17.199
`
`30-360
`
`millilitre
`
`5-2-2. Plate-count methods
`5-2-2-1. Pour-plate method
`Prepare the sample using a method that has been shown to be
`suitable as described in section 4. Prepare for each medium at
`least 2 Petri dishes for each level of dilution. Incubate
`the plates
`of casein soya bean digest agar at 30-35 C for 3-5 days and
`agar at 20-25 C for 5-7 days.
`the plates of Sabouraud-dextrose
`Select
`the plates corresponding to a given dilution and showing
`the highest number of colonies less than 250 for TAMC and 50
`for TYMC. Take the arithmetic mean per culture medium of
`the number of CFU per gram or per
`the counts and calculate
`of product.
`5-2-2-2. Surface-spread method
`Prepare the sample using a method that has been shown to be
`suitable as described in section 4. Prepare at least 2 Petri dishes
`for each medium and each level of dilution. For incubation
`and
`of the number of CFU proceed as described
`for the
`calculation
`pour-plate method.
`
`5-2-3. Most-probable-number method
`Prepare and dilute the sample using a method that has been
`shown to be suitable as described in section 4. Incubate
`tubes at 30-35 C for 3-5 days. Subculture
`
`if necessary using
`shown to be suitable. Record for each level
`the procedure
`of dilution the number of tubes showing microbial growth.
`Determine the most probable number of micro-organisms per
`gram or millilitre
`of the product to be examined from Table
`
`all
`
`2.6.12.-3.
`
`OF THE RESULTS
`5-3. INTERPRETATION
`The total aerobic microbial count TAMC is considered to be
`equal to the number of CFU found using casein soya bean digest
`agar if colonies of fungi are detected on this medium they are
`counted as part of the TAMC. The total combined yeasts/mould
`count TYMC is considered to be equal to the number of CFU
`agar
`if colonies of bacteria
`found using Sabouraud-dextrose
`are detected on this medium they are counted as part of the
`
`3
`
`3
`
`3
`
`3
`
`3
`
`3
`
`3
`
`3
`
`3
`
`3
`
`1
`
`2
`
`2
`
`2
`
`2
`
`3
`
`3
`
`3
`
`3
`
`2
`
`3
`
`0
`
`1
`
`2
`
`3
`
`0
`
`1
`
`2
`
`3
`
`120
`
`160
`
`93
`
`150
`
`210
`
`290
`
`240
`
`460
`
`30.380
`
`18360
`
`30-380
`
`30400
`
`90-990
`
`40 990
`
`90-1980
`
`1100
`
`200-4000
`
`1100
`
`The amount to be tested may be reduced for active substances
`that will be formulated in the following conditions the amount
`tablet capsule injection is less than
`per dosage unit e.g.
`to 1 mg or the amount per gram or millilitre
`or equal
`for
`
`166
`
`See the information section
`
`on general monographs cover pages
`
`LUPIN068842
`
`Page 6
`
`

`
`EUROPEAN PHARMACOPOEIA
`
`7.0
`
`2.6.13. Test
`
`for specified micro-organisms
`
`TYMC. When the TYMC is expected to exceed the acceptance
`criterion due to the bacterial growth Sabouraud-dextrose
`agar
`antibiotics may be used.
`the count
`is carried out
`containing
`If
`by the MPN method the calculated
`value is the TAMC.
`When an acceptance criterion for microbiological quality is
`is interpreted as follows
`prescribed
`- 101 CFU maximum acceptable count
`- 102 CFU maximum acceptable count
`- 103 CFU maximum acceptable count
`The recommended
`solutions and media are described in general
`
`20
`200
`2000 and so forth.
`
`it
`
`chapter 2.6.13.
`
`04/201020613
`
`2.6.13. MICROBIOLOGICAL
`EXAMINATION OF NON-STERILE
`PRODUCTS TEST FOR SPECIFIED
`MICRO-ORGANISMS2
`
`1.
`
`INTRODUCTION
`
`The tests described hereafter will allow determination of the
`absence or limited occurrence of specified micro-organisms that
`may be detected under the conditions described.
`
`The tests are designed primarily to determine whether
`a substance or preparation complies with an established
`for microbiological quality. When used for such
`specification
`purposes follow the instructions given below including the
`number of samples to be taken and interpret
`the results as
`stated below.
`
`Alternative microbiological procedures including automated
`methods may be used provided
`to the
`that their equivalence
`Pharmacopoeia method
`has been demonstrated.
`
`2. GENERAL
`
`PROCEDURES
`
`The preparation of samples is carried out as described in general
`chapter 2.6.12.
`
`If
`
`has antimicrobial activity this
`the product to be examined
`is insofar as possible removed or neutralised as described
`general chapter 2.6.12.
`
`in
`
`substances are used for sample preparation
`If surface-active
`their absence of toxicity for micro-organisms and their
`compatibility with inactivators used must be demonstrated
`described in general chapter 2.6.12.
`
`as
`
`AND INHIBITORY PROPERTIES
`3. GROWTH-PROMOTING
`OF THE MEDIA SUITABILITY OF THE TEST AND NEGATIVE
`CONTROLS
`
`to detect micro-organisms in the presence
`The ability of the test
`to be tested must be established. Suitability must
`
`of the productuct
`be confirmed if a change in testing performance or the product
`the outcome of the test is introduced.
`which may affect
`3-1. PREPARATION OF TEST STRAINS
`Use standardised
`stable suspensions of test strains or prepare
`them as stated below. Seed lot culture maintenance
`techniques
`seed-lot systems are used so that the viable micro-organisms
`used for inoculation are not more than 5 passages removed
`from the original master seed-lot.
`
`strains separately
`
`3-1-1. Aerobic micro-organisms. Grow each of the bacterial
`test
`in casein soya bean digest broth or on casein
`soya bean digest agar at 30-35 C for 18-24 h. Grow the test
`strain for Candida albicans separately
`broth at 20-25 C for 2-3 days.
`agar or in Sabouraud-dextrose
`aureus such as ATCC 6538 NCIMB 9518
`- Staphylococcus
`CIP 4.83 or NBRC 13276
`- Pseudomonas aeruginosa such as ATCC 9027 NCIMB 8626
`CIP 82.118 or NBRC 13275
`
`on Sabouraud-dextrose
`
`- Escherichia coli such as ATCC 8739 NCIMB 8545
`CIP 53.126 or NBRC 3972
`- Salmonella enterica subsp. enterica serovar Typhimurium
`such as ATCC 14028 or as an alternative Salmonella
`enterica subsp. enterica serovar Abony such as
`NBRC 100797 NCTC 6017 or CIP 80.39
`- Candida albicans such as ATCC 10231 NCPF 3179 IP 48.72
`or NBRC 1594.
`
`solution pH 7.0 or
`Use buffered sodium chloride-peptone
`phosphate buffer solution pH 7.2 to make test suspensions.
`Use
`the suspensions within 2 h or within 24 h if stored at 2-8 C.
`
`such as ATCC
`3-1-2. Clostridia. Use Clostridium sporogenes
`11437 NBRC 14293 NCIMB 12343 CIP 100651 or ATCC
`19404 NCTC 532 or CIP 79.03 or NBRC 14293. Grow the
`clostridial test strain under anaerobic conditions in reinforced
`medium for clostridia at 30-35 C for 24-48 h. As an alternative
`to preparing and then diluting down a fresh suspension of
`vegetative cells of Cl. sporogenes a stable spore suspension
`used for test inoculation. The stable spore suspension may be
`maintained at 2-8 C for a validated
`period.
`3-2. NEGATIVE CONTROL
`To verify testing conditions a negative control
`is performed
`using the chosen diluent
`in place of the test preparation. There
`must be no growth of micro-organisms. A negative control
`is also performed when testing the products
`as described in
`section 4. A failed negative control requires an investigation.
`3-3. GROWTH PROMOTIONAND INHIBITORY PROPERTIES
`OF THE MEDIA
`Test each batch of ready prepared medium and each batch of
`medium prepared either from dehydrated medium or from
`
`is
`
`in
`
`ingredients.
`Verify suitable properties of relevant media as described
`Table 2.6.11-1.
`Test for growth promoting properties liquid media inoculate
`a portion of the appropriate medium with a small number
`not more than 100 CFU of the appropriate micro-organism.
`Incubate at the specified temperature for not more than the
`shortest period of time specified in the test. Clearly visible
`growth of the micro-organism comparable
`to that previously
`obtained with a previously tested and approved
`batch of
`medium occurs.
`Test for growth promoting properties solid media perform
`the surface-spread method inoculating each plate with a
`small number not more than 100 CFU of the appropriate
`micro-organism. Incubate
`at the specified temperature for not
`more than the shortest period of time specified in the test.
`Growth of the micro-organism comparable
`to that previously
`obtained with a previously tested and approved
`batch of
`medium occurs.
`
`the
`
`for inhibitory properties liquid or solid media inoculate
`appropriate medium with at least 100 CFU of the appropriate
`micro-organism. Incubate
`at the specified temperature for not
`less than the longest period of time specified in the test. No
`growth of the test micro-organism occurs.
`
`Test for indicative properties perform the surface-spread
`method inoculating each plate with a small number not more
`than 100 CFU of the appropriat