I
`
`SEVENTH EDITION
`Volume 1
`
`Published in accordance with the
`Convention on the Elaboration of a European Pharmacopoeia
`(European Treaty Series No. 50)
`
`* t<~'t!;:., Dicedocate foe the
`
`Quality of Medicines & HealthCare
`
`Council of Europe
`Strasbourg
`
`LUPIN069102
`
`SENJU EXHIBIT 2295
`LUPIN v. SENJU
`IPR2015-01099
`
`Page 1 of 18
`
`

`
`The European Pharmacopoeia is published by the Directorate for the Quality of Medicines &
`Health Care of the Council of Europe (EDQM).
`
`©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
`
`LUPIN069103
`
`Page 2 of 18
`
`

`
`EUROPEAN PHARMACOPOEIA 7.0
`
`2.6.12. Microbial enumeration tests
`
`the animal from loss of body heat and maintain it so that
`the rectal temperature remains within physiological limits.
`Introduce a cannula into the trachea. Insert a cannula filled
`with a heparinised 9 gjL solution of sodium chloride into the
`common carotid artery and connect it to a device capable of
`giving a continuous record of the blood pressure. Insert into the
`femoral vein another cannula, filled with a heparinised 9 gjL
`solution of sodium chloride, through which can be injected the
`solutions of histamine and of the substance to be examined.
`Determine the sensitivity of the animal to histamine by injecting
`intravenously at regular intervals, doses of histamine solution R
`corresponding to 0.1 j.Jg and 0.15 j.Jg of histamine base per
`kilogram of body mass. Repeat the lower dose at least 3 times.
`Administer the second and subsequent injections not less than
`1 min after the blood pressure has returned to the level it
`was at immediately before the previous 'injection. The animal
`is used for the test only if a readily discernible decrease in
`blood pressure that is constant for the lower dose is obtained
`and if the higher dose causes greater responses. Dissolve the
`substance to be examined in sufficient of a 9 gjL solution
`of sodium chloride or other prescribed solvent, to give the
`prescribed concentration. 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
`to be examined and, finally, 1.0 mL of histamine solution R.
`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.
`If 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 fails the test if the mean
`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
`causes a greater depressor response than the concluding dose
`of the histamine solution. The test animal must not be used in
`another test for depressor substances if the second criterion
`applies or if the response to the high dose of histamine given
`after the administration of the substance to be examined is
`less than the mean response to the low doses of histamine
`previously injected.
`·
`
`07/2010:20612
`
`2.6.12. MICROBIOLOGICAL
`EXAMINATION OF NON-STERILE
`PRODUCTS: MICROBIAL ENUMERATION
`TESTS(l)
`
`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
`specification for microbiological quality. When used for such
`purposes follow the instructions given below, including the
`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 that their equivalence to the
`Pharmacopoeia method has been demonstrated.
`
`2.GENERALPROCEDURES
`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 the product to be examined has antimicrobial activity, this is
`insofar as possible removed or neutralised. If inactivators are
`used for this purpose, their efficacy and their absence of toxicity
`for micro-organisms must be demonstrated.
`If surface-active substances are used for sample preparation,
`their absence of toxicity for micro-organisms and their
`compatibility with inactivators used must be demonstrated.
`
`3. ENUMERATION METHODS
`Use the membrane filtration method or the plate-count methods,
`as prescribed. The most-probable-number (MPN) method is
`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
`judge compliance with the specification. The suitability of the
`method chosen must be established.
`
`4. GROWTH PROMOTION TEST, SUITABILITY OF THE
`COUNTING METHOD AND NEGATIVE CONTROLS
`4-1. GENERAL CONSIDERATIONS
`The ability of the test to detect micro-organisms in the presence
`of product to be tested must be established.
`Suitability must be confirmed if a change in testing performance,
`or the product, which may affect the outcome of the test is
`introduced.
`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
`and fungal test strains separately as described in Table 2.6.12.-1.
`Use buffered sodium chloride-peptone solution pH 7.0 or
`phosphate buffer solution pH 7.2 to make test suspensions; to
`suspend A. brasiliensis spores, 0.05 per cent of polysorbate 80
`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
`preparing and then diluting a fresh suspension of vegetative
`cells of A. brasiliensis or B. subtilis, a stable spore suspension
`is prepared and then an appropriate volume of the spore
`suspension is used for test inoculation. The stable spore
`suspension may be maintained at 2-8 oc for a validated period
`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 2.6.12.-1,
`using a separate portion/plate of medium for each. Inoculate
`plates of Sabouraud-dextrose agar with a small number (not
`
`(1) This chapter has undergone pharmacopoeial harmonisation. See chapter 5.8. Pharmacopoeial harmonisation.
`
`General Notices (1) apply to all monographs and other texts
`
`163
`
`LUPIN069104
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`Page 3 of 18
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`

`
`2.6.12. l\iic:robial enumeration tests
`
`EUROPEAN PHARMACOPOEIA 7.0
`
`Micrcrorganism
`
`Table 2.6.12.-1. -Preparation and use oftest micro-organisms
`Suitability of counting method in the
`Preparation of test
`Growth promotion
`strain
`presence of the product
`Total aerobic microbial
`Total yeasts and
`count
`moulds count
`Casein soya bean digest
`agar/MPN casein soya
`bean digest broth
`:;; 100 CFU
`30-35 "C
`:s; 3 days
`
`Total yeasts and
`moulds count
`
`Total aerobic microbial
`count
`Staphylococcus aureus Casein soya bean digest Casein soya bean digest
`agar or casein soya
`agar and casein soya
`such as:
`bean digest broth
`bean digest broth
`ATCC 6538
`30-35 oc
`:s; 100 CFU
`NCIMB 9518
`18-24 h
`30-35 "C
`ClP 4.83
`:s; 3 days
`NBRC 13276
`Pseudomonas
`aeruginosa
`such as:
`ATCC 9027
`NCIMB 8626
`ClP 82.118
`NBRC 13275
`Bacillus subtilis
`such as:
`ATCC 6633
`NCIMB 8054
`ClP 52.62
`NBRC 3134
`Candida a/bicans
`such as:
`ATCC 10231
`NCPF 3179
`IP 48.72
`NBRC 1594
`Aspergillus brasiliensis
`such as:
`ATCC 16404
`IM1149007
`lP 1431.83
`NBRC 9455
`
`Casein soya bean digest Casein soya bean digest
`agar or casein soya
`agar and casein soya
`bean digest broth
`bean digest broth
`30-35 oc
`:s; 100 CFU
`30-35 oc
`18-24 h
`53 days
`
`Casein soya bean digest Casein soya bean digest
`agar or casein soya
`agar and casein soya
`bean digest broth
`bean digest broth
`30-35 oc
`:> 100 CFU
`30-35 oc
`18-24 h
`:> 3 days
`
`Sabouraud-dextrose
`agar or Sabouraud-
`dextrose broth 20-25 o C
`2-3 days
`
`Sabouraud-dextrose
`agar or potato-dextrose
`agar
`20-25 oc
`5-7 days, or until good
`sporulation is achieved
`
`Casein soya bean
`digest agar
`,; 100 CFU
`30-35 oc
`:> 5 days
`
`Casein soya bean
`digest agar
`,; 100 CFU
`30-35 oc
`:> 5 days
`
`more than 100 CFU) of the micro-organisms indicated in Table
`2.6.12.-1, using a separate plate of medium for each. Incubate
`in the conditions described in Table 2.6.12.-1.
`For solid media, growth obtained must not differ by a factor
`greater than 2 from the calculated value for a standardised
`inoculum. For a freshly prepared inoculum, growth of the
`micro-organisms comparable to that previously obtained with
`a previously tested and approved batch of medium occurs.
`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 characteristics of the
`product to be tested. If none of the procedures described below
`can be demonstrated to be satisfactory, an alternative procedure
`must be developed.
`Water-soluble products. Dissolve or dilute (usually a 1 in 10
`dilution is prepared) the product to be examined in buffered
`sodium chloride-peptone solution pH 7.0, phosphate buffer
`solution pH 7.2 or casein soya bean digest broth. If necessary,
`adjust to pH 6-8. Further dilutions, where necessary, are
`prepared with the same diluent.
`Non-fatty products insoluble in water. Suspend the product
`to be examined (usually a 1 in 10 dilution is prepared) in
`buffered sodium chloride-peptone solution pH 7_0, phosphate
`buffer solution pH 7.2 or casein soya bean digest broth. A
`surface-active agent such as 1 gjL of polysorbate 80 may be
`added to assist the suspension of poorly wettable substances. If
`necessary, adjust to pH 6-8. Further dilutions, where necessary,
`are prepared with the same diluent.
`Fatty products. Dissolve in isopropyl myristate, sterilised by
`filtration or mix the product to be examined with the minimum
`necessary quantity of sterile polysorbate SO or another
`
`Casein soya bean digest
`agar/MPN casein soya
`bean digest broth
`,; 100 CFU
`30-35 oc
`53 days
`
`Casein soya bean digest
`agar/MPN casein soya
`bean digest broth
`,; 100 CFU
`30-35 oc
`,; 3 days
`
`Casein soya bean
`digest agar
`,; 100 CFU
`30-35 oc
`55 days
`MPN: not applicable
`Casein soya bean
`digest agar
`,; 100 CFU
`30-35 oc
`5 5 days
`MPN: not applicable
`
`Sabouraud-dextrose
`agar
`,; 100 CFU
`20-25 oc
`,; 5 days
`
`Sabouraud-dextrose
`agar
`:s; 100 CFU
`20-25 oc
`:> 5 days
`
`Sabouraud-dextrose
`agar
`:;; 100 CFU
`20-25 oc
`55 days
`
`Sabouraud-dextrose
`agar
`:;; 100 CFU
`20-25 oc
`55 days
`
`I
`non-inhibitory sterile surface-active agent, heated if necessary to
`not more than 40 o C, or in exceptional cases to not more than
`45 o C. Mix carefully and if necessary maintain the temperature
`in a water-bath. Add sufficient of the pre-warmed 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
`product into a membrane filter apparatus or a sterile container
`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 lecithin.
`Shake the preparation vigorously for at least 30 min.
`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
`
`To demonstrate 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
`protocols must be developed. If inhibition of growth by the
`sample cannot otherwise be avoided, the aliquot of the microbial
`
`164
`
`See the information section on general monographs (cover pages)
`
`LUPIN069105
`
`Page 4 of 18
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`

`
`EUROPEAN PHARMACOPOEIA 70
`
`2.6.12. Microbial enumeration tests
`
`suspension may be added after neutralisation, dilution or
`filtration.
`4-5-3. Neutralisation/removal of antimicrobial activity. The
`number of micro-organisms recovered from the prepared
`sample diluted as described in 4-5-2 and incubated following
`the procedure described in 4-5-4, is compared to the number of
`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 2.6.12.-2).
`They may be added to the chosen diluent or the medium
`preferably before sterilisation. If used, their efficacy and their
`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
`
`Glutaraldehyde, mercurials
`
`Potential neutralising
`method
`Sodium hydrogensulfite
`(sodium bisulfite)
`
`Phenolics, alcohol, aldehydes, sorbate
`
`Aldehydes
`
`Quaternary Ammonium Compounds
`(QACs), parahydroxybenzoates (parabens),
`bis-biguanides
`QACs, iodine, parabens
`
`Mercurials
`
`Dilution
`
`Glycine
`
`Lecithin
`
`Polysorbate
`
`Thioglycollate
`
`Mercurials, halogens, aldehydes
`
`Thiosulfate
`
`EDTA (edetate)
`
`Mg2• or Ca'· 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, it 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
`latter are not representative. Then, perform the test with the
`highest dilution factor compatible with microbial growth and
`the specific acceptance criterion.
`4-5-4. Recovery of micro-organism in the presence of product.
`For each of the micro-organisms listed, separate tests are
`performed. Only micro-organisms of the added test strain are
`counted.
`
`4-5-4-1. Membrane filtration. Use membrane filters having a
`nominal pore size not greater than 0.45 !Jm. The type of filter
`material is chosen such that the bacteria-retaining efficiency is
`not affected by the components of the sample to be investigated.
`For each of the micro-organisms listed, one membrane filter
`is used.
`
`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,
`or less if large numbers of CFU are expected) to the membrane
`filter, filter immediately and rinse the membrane filter with an
`appropriate volume of diluent.
`
`For the determination of total aerobic microbial count (TAM C),
`transfer the membrane filter to the surface of casein soya
`bean digest agar. For the determination of total combined
`
`yeasts/moulds count (TYMC), transfer the membrane to the
`surface of Sabouraud-dextrose agar. Incubate the plates as
`indicated in Table 2.6.12.-1. Perform the counting.
`4-5-4-2. Plate-count methods. Perform plate-count methods at
`least in duplicate for each medium and use the mean count of
`the result.
`4-5-4-2-1. Pour-plate method
`For Petri dishes 9 em in diameter, add to the dish I 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 2.6.12.-1, at
`least 2 Petri dishes are used. Incubate the plates as indicated
`in Table 2.6.12.-1. Take the arithmetic mean of the counts
`per medium and calculate the number of CFU in the original
`inoculum.
`4-5-4-2-2. Surface-spread method
`For Petri dishes 9 em in diameter, add 15-20 mL of casein soya
`bean digest agar or Sabouraud-dextrose agar at about 45 o C to
`each Petri dish and allow to solidify. If larger Petri dishes are
`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 less
`than 0.1 mL of the sample prepared as described under 4-5-1
`to 4-5-3 over the surface of the medium. Incubate and count
`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
`is available. If the use of the method is justified, proceed as
`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, a
`surface-active agent such as polysorbate 80 or an inactivator of
`antimicrobial agents may be added to the medium. Thus, if 3
`levels of dilution are prepared, 9 tubes are inoculated.
`Incubate all tubes at 30-35 oc for not more than 3 days. If
`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 of
`the product to be examined from Table 2.6.12.-3.
`4-6. RESULTS AND INTERPRETATION
`When verifying the suitability of. the membrane filtration
`method or the plate-count method, a mean count of any of the
`test organisms not differing by a factor greater than 2 from
`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 limits of the results obtained
`with the control.
`If the above criteria cannot be met for one or more of the
`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
`to be examined taken with the precautions referred to above.
`For fluids or solids in aerosol form, sample 10 containers. For
`transdermal patches, sample 10 patches.
`
`General Notices (1) apply to all monographs and other texts
`
`165
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`

`
`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 eacb set
`:-.lumber of grams or millilitres of
`product per tube
`
`0.1
`
`0.01
`
`0.001
`
`MPN per
`gram or per
`millilitre of
`product
`
`95 per cent
`confidence
`limits
`
`0
`
`0
`
`0
`
`0
`
`0
`
`0
`
`1
`
`1
`
`1
`
`0
`
`0
`
`1
`
`1
`
`2
`
`3
`
`0
`
`0
`
`0
`
`0
`
`1
`
`0
`
`1
`
`0
`
`0
`
`0
`
`1
`
`2
`
`< 3
`
`3
`
`3
`
`6.1
`
`6.2
`
`9.4
`
`3.6
`
`7.2
`
`11
`
`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
`
`4-35
`
`1
`
`1
`
`1
`
`1
`
`1
`
`2
`
`2
`
`2
`
`2
`
`2
`
`1
`
`1
`
`2
`
`2
`
`3
`
`0
`
`0
`
`0
`
`1
`
`1
`
`0
`
`1
`
`0
`
`1
`
`0
`
`0
`
`1
`
`2
`
`0
`
`1
`
`2
`
`7.4
`
`11
`
`11
`
`15
`
`16
`
`9.2
`
`14
`
`20
`
`15
`
`.20
`
`27
`
`1.3-20
`
`4-35
`
`4-35
`
`5-38
`
`5-38
`
`1.5-35
`
`4-35
`
`5-38
`
`4-38
`
`5-38
`
`9-94
`
`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
`present in 10 dosage units or 10 g or 10 mL of the product.
`For materials used as active substances where sample quantity
`is limited or batch size is extremely small (i.e. Jess than 1000 mL
`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 1 unit if the size is less than 100.
`Select the sample(s) at random from the bulk material or from
`the available containers of the preparation. To obtain the
`required quantity, mix the contents of a sufficient number of
`containers to provide the sample.
`5-2. EXAMINATION OF THE PRODUCT
`5-2-1. Membrane filtration
`Use a filtration apparatus designed to allow the transfer of the
`filter to the medium. Prepare the sample using a method that
`has been shown suitable as described in section 4 and transfer
`the appropriate amount to each of 2 membrane filters and filter
`immediately. Wash each filter following the procedure shown
`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 agar. Incubate the plate of casein
`soya bean digest agar at 30-35 o C for 3-5 days and the plate of
`Sabouraud-dextrose agar at 20-25 oc for 5-7 days. Calculate the
`number of CFU per gram or per millilitre of product.
`When examining b·ansdermal patches, filter 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.
`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 o C for 3-5 days and
`the plates of Sabouraud-dextrose agar at 20-25 oc for 5-7 days.
`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 counts and calculate the number of CFU per gram or per
`millilitre 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
`calculation of the number of CFU proceed as described for the
`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 all
`tubes at 30-35 oc for 3-5 days. Subculture if necessary, using
`the procedure shown to be suitable. Record for each level
`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
`2.6.12.-3.
`5-3. INTERPRETATION OF THE RESULTS
`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
`found using Sabouraud-dextrose agar; if colonies of bacteria
`are detected on this medium, they are counted as part of the
`
`3
`
`3
`
`3
`
`3
`
`3
`
`3
`
`3
`
`3
`
`3
`
`3
`
`1
`
`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
`
`llOO
`
`> 1100
`
`30-380
`
`18-360
`
`30-380
`
`30-400
`
`90-990
`
`40-990
`
`90-1980
`
`200-4000
`
`The amount to be tested may be reduced for active substances
`that will be formulated in the following conditions: the amount
`per dosage unit (e.g. tablet, capsule, injection) is less than
`or equal to 1 mg or the amount per gram or millilitre (for
`
`166
`
`See the information section on general monographs (cover pages)
`
`LUPIN069107
`
`2
`
`2
`
`2
`
`2
`
`2
`
`2
`
`3
`
`3
`
`3
`
`3
`
`3
`
`1
`
`2
`
`2
`
`2
`
`3
`
`3
`
`0
`
`0
`
`0
`
`1
`
`1
`
`0
`
`1
`
`2
`
`0
`
`1
`
`0,
`
`1
`
`2
`
`0
`
`1
`
`21
`
`28
`
`35
`
`29
`
`36
`
`23
`
`38
`
`64
`
`43
`
`75
`
`5-40
`
`9-94
`
`9-94
`
`9-94
`
`9-94
`
`5-94
`
`9-104
`
`16-181
`
`9-181
`
`17-199
`
`30-360
`
`Page 6 of 18
`
`

`
`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
`containing antibiotics may be used. If the count is carried out
`by the MPN method the calculated value is the TAMC.
`When an acceptance criterion for microbiological quality is
`prescribed it is interpreted as follows:
`101 CFU: maximum acceptable count= 20;
`_ 102 CFU: maximum acceptable count = 200;
`103 CFU: maximum acceptable count= 2000, and so forth.
`-
`The recommended solutions and media are described in general
`chapter 2.6.13.
`
`04/2010:20613
`
`2.6.13. MICROBIOLOGICAL
`EXAMINATION OF NON-STERILE
`PRODUCTS: TEST FOR SPECIFIED
`MICRO-ORGANISMSr2J
`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
`specification for microbiological quality. When used for such
`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 that their equivalence to the
`Pharmacopoeia method has been demonstrated.
`
`2. GENERAL PROCEDURES
`The preparation of samples is carried out as described in general
`chapter 2.6.12.
`If the product to be examined has antimicrobial activity, this
`is insofar as possible removed or neutralised as described in
`general chapter 2.6.12.
`If surface-active substances are used for sample preparation,
`their absence of toxicity for micro-organisms and their
`compatibility with inactivators used must be demonstrated as
`described in general chapter 2.6.12.
`
`3. GROWTH-PROMOTING AND INHIBITORY PROPERTIES
`OF THE MEDIA, SUITABILITY OF THE TEST AND NEGATIVE
`CONTROLS
`The ability of the test to detect micro-organisms in the presence
`of the product to be tested must be established. Suitability must
`be confirmed if a change in testing performance, or the product,
`which may affect the outcome of the test is introduced.
`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.
`3-1-1. Aerobic micro-organisms. Grow each of the bacterial test
`strains separately in casein soya bean digest broth or on casein
`soya bean digest agar at 30-35 oc for 18-24 h. Grow the test
`strain for Candida albicans separately on Sabouraud-dextrose
`agar or in Sabouraud-dextrose broth at 20-25 o C for 2-3 days.
`Staphylococcus aureus such as ATCC 6538, NCIMB 9518,
`CIP 4.83 or NBRC 13276;
`Pseudomonas aeruginosa such as ATCC 9027, NCIMB 8626,
`CIP 82.118 or NBRC 13275;
`
`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.
`Use buffered sodium chloride-peptone solution pH 7.0 or
`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 oc.
`3-1-2. Clostridia. Use Clostridium sporogenes such as ATCC
`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 oc 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 is
`used for test inoculation. The stable spore suspension may be
`maintained at 2-8 oc 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 PROMOTION AND INHIBITORY PROPERTIES
`OF THE MEDIA
`Test each batch of ready-prepared medium and each batch of
`medium prepared either from dehydrated medium or from
`ingredients.
`Verify suitable properties of relevant media as described in
`Table 2.6.13.-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 n