EUROPEAN PHARMACOPOEIA 10.0 5.1.10.

Guidelines for using the test for bacterial endotoxins

01/2009:50109 methods may not be sufficiently sensitive or reliable enough to

provide unequivocal evidence that 2 isolates are from the same
source. More sensitive tests, for example molecular typing
with RNA/DNA homology, may be necessary to determine
that micro-organisms are clonally related and have a common
5.1.9. GUIDELINES FOR USING THE origin.
TEST FOR STERILITY
The purpose of the test for sterility (2.6.1), as that of all 07/2016:50110
pharmacopoeial tests, is to provide an independent control
analyst with the means of verifying that a particular material
meets the requirements of the European Pharmacopoeia. A
manufacturer is neither obliged to carry out such tests nor
precluded from using modifications of, or alternatives to, the
stated method, provided he is satisfied that, if tested by the
5.1.10. GUIDELINES FOR USING THE
official method, the material in question would comply with TEST FOR BACTERIAL ENDOTOXINS
the requirements of the European Pharmacopoeia.
1. INTRODUCTION
PRECAUTIONS AGAINST MICROBIAL Endotoxins from gram-negative bacteria are the
CONTAMINATION most common cause of toxic reactions resulting from
Aseptic conditions for performance of the test can be achieved contamination of pharmaceutical products with pyrogens ;
using, for example, a class A laminar-air-flow cabinet located their common pyrogenic activity is much higher than that
within a class B clean room, or an isolator of other known pyrogenic substances. These endotoxins
are lipopolysaccharides. Although there are a small
GUIDANCE TO MANUFACTURERS number of pyrogens that possess a different structure,
The level of assurance provided by a satisfactory result of a the conclusion is generally justified that the absence of
test for sterility (the absence of contaminated units in the bacterial endotoxins in a substance or product implies the
sample) as applied to the quality of the batch is a function of absence of pyrogenic components, provided the presence of
the homogeneity of the batch, the conditions of manufacture non-endotoxin pyrogenic substances can be ruled out. The
and the efficiency of the adopted sampling plan. Hence for monocyte-activation test (2.6.30) is a suitable method to
the purpose of this text a batch is defined as a homogeneous use to rule out the presence of non-endotoxin pyrogens in
collection of sealed containers prepared in such a manner that substances or products.
the risk of contamination is the same for each of the units The presence of endotoxins in a substance or product may
contained therein. be masked by factors interfering with the reaction between
In the case of terminally sterilised products, physical proofs, the endotoxins, the test reagents and the amoebocyte lysate.
biologically based and automatically documented, showing Also, the ability to detect endotoxins may be affected by
correct treatment throughout the batch during sterilisation are storage conditions or storage time. Hence, the analyst who
of greater assurance than the sterility test. The circumstances wishes to implement a test for bacterial endotoxins or to
in which parametric release may be considered appropriate replace the pyrogen test by a test for bacterial endotoxins has
are described under 5.1.1. Methods of preparation of sterile to demonstrate that a valid test can be carried out on the
products. The method of media-fill runs may be used to substance or product concerned ; this may entail a procedure
evaluate the process of aseptic production. Apart from that, for removing interference.
the sterility test is the only analytical method available for As indicated in general chapter 2.6.14. Bacterial endotoxins,
products prepared under aseptic conditions and furthermore information must be available on the following 2 aspects
it is, in all cases, the only analytical method available to the before a test on a sample can be regarded as valid.
authorities who have to examine a specimen of a product for – The suitability of the material to be used for the test has
sterility. to be established. The absence of endotoxins in the water
The probability of detecting micro-organisms by the test for for BET (water for bacterial endotoxins test) and in the
sterility increases with their number present in the sample other reagents and consumables must be assured and the
tested and varies according to the readiness of growth of sensitivity of the amoebocyte lysate must be checked to
micro-organism present. The probability of detecting very confirm the sensitivity declared by the manufacturer.
low levels of contamination even when it is homogenous – As the substance or product to be examined may interfere
throughout the batch is very low. The interpretation of the with the test, the sensitivity of the amoebocyte lysate is
results of the test for sterility rests on the assumption that determined in the presence and in the absence of the
the contents of every container in the batch, had they been substance or product to be examined. There must be no
tested, would have given the same result. Since it is manifest difference between the 2 sensitivity values.
that every container cannot be tested, an appropriate sampling
plan should be adopted. In the case of aseptic production, it is General chapter 2.6.14. Bacterial endotoxins indicates
recommended to include samples filled at the beginning and methods for removing interfering factors ; in the case of
at the end of the batch and after significant intervention. interference, another test must be carried out after such a
method has been applied to check whether the interference
OBSERVATION AND INTERPRETATION OF RESULTS has indeed been neutralised or removed.
Conventional microbiological/biochemical techniques are This general chapter explains the reasons for the requirements
generally satisfactory for identification of micro-organisms in the test for bacterial endotoxins, then deals with reading
recovered from a sterility test. However, if a manufacturer and interpretation of the results.
wishes to use condition (d) as the sole criterion for invalidating Replacement of the rabbit pyrogen test required in a
a sterility test, it may be necessary to employ sensitive typing pharmacopoeial monograph by an amoebocyte lysate test, or
techniques to demonstrate that a micro-organism isolated by other methods such as the monocyte-activation test or a
from the product test is identical to a micro-organism isolated test using recombinant factor C reagent as a replacement for
from the test materials and/or the testing environment. While the amoebocyte lysate, constitutes the use of an alternative
routine microbiological/biochemical identification techniques method of analysis and hence requires demonstration that
can demonstrate that 2 isolates are not identical, these the method is appropriate for the given substance or product

General Notices (1) apply to all monographs and other texts 639
5.1.10. Guidelines for using the test for bacterial endotoxins EUROPEAN PHARMACOPOEIA 10.0

and gives a result consistent with that obtained with the K = threshold pyrogenic dose of endotoxin per
prescribed method as described in the General Notices (see kilogram of body mass ;
also section 12). M = maximum recommended bolus dose of product
per kilogram of body mass.
The prescribed method for bacterial endotoxins may be stated
in the monograph on a given substance or product. The use of When the product is to be injected at frequent intervals
a method other than the method prescribed in the monograph or infused continuously, M is the maximum total dose
is considered as the use of an alternative method. Where no administered per hour.
method is stated, any of methods A to F of general chapter The endotoxin limit depends on the product and its route of
2.6.14. Bacterial endotoxins can be used. administration and may be stated in the monograph. Values
for K are suggested in Table 5.1.10.-1.
2. METHOD AND ACCEPTANCE CRITERIA For other routes, the acceptance criterion for bacterial
2-1. METHODS AND PRECAUTIONS TO BE TAKEN endotoxins is generally determined on the basis of results
The addition of endotoxins to amoebocyte lysate may result obtained during the development of the preparation.
in turbidity, precipitation or gelation (gel-clot); initially only
the gel-clot method was used in the Pharmacopoeia as an Table 5.1.10.-1
evaluation criterion in the test for bacterial endotoxins. The Route of administration K
advantage was the simplicity of basing the decision to pass or
Intravenous 5.0 IU of endotoxin per kilogram
fail the substance or product to be examined on the absence of body mass
or presence of a gel-clot, visible with the naked eye. The
quantitative methods C, D, E and F were developed later : they Intravenous for radiopharmaceuticals 2.5 IU of endotoxin per kilogram
of body mass
require more instrumentation, but they are easier to automate
for the regular testing of large numbers of samples of the same Intrathecal 0.2 IU of endotoxin per kilogram
of body mass
substance or product.
Parenteral formulations administered 100 IU/m2
Endotoxins may be adsorbed onto the surface of tubes per square metre of body surface
or pipettes made from certain plastics or types of glass.
Interference may appear due to the release of substances from 2-4. CONSIDERATIONS WHEN ESTABLISHING AN
plastic materials. Hence, the materials used must be checked. ENDOTOXIN LIMIT FOR A SPECIFIC SUBSTANCE OR
2-2. ENDOTOXIN LIMIT CONCENTRATION PRODUCT
The endotoxin limit for a substance or product is established
The decision to use the test for bacterial endotoxins as a limit
with consideration of the following aspects.
test implies firstly that an endotoxin limit concentration must
be defined for the substance or product to be examined, and Calculated endotoxin limit. The endotoxin limit is calculated
secondly that the objective of the test is to know whether the as described in section 2-3. This represents a safety limit not
endotoxin concentration in the sample to be examined is below to be exceeded if the product is to be administered to humans.
or above this limit. The quantitative methods C, D, E and F Limit prescribed in an individual substance monograph.
make it possible to determine the endotoxin concentration The limit stated in an individual substance monograph
in the sample to be examined, but for compliance with the frequently reflects what is achievable in a controlled
Pharmacopoeia and in routine quality control the final production environment. The limit prescribed in a
question is whether or not this concentration exceeds a monograph can therefore be lower than the calculated
defined limit. endotoxin limit. However a manufacturer may specify a limit
The dose of the substance or product to be examined that is more stringent than that stated in the monograph.
must be taken into account in setting the endotoxin limit Process capability. The capability of the process to reduce or
concentration : the limit is set so as to ensure that, as long remove bacterial endotoxins during manufacture might result
as the endotoxin concentration in the substance or product in lower endotoxin limits for specific processes.
remains below this limit, even the maximal dose administered
by the intended route per hour does not contain sufficient Additional safety requirements. Precautions are taken
endotoxin to cause a toxic reaction. in consideration of patient population (such as paediatric
use, malnourished or cachectic patients, etc.), specific local
When the endotoxin concentration in the substance or product requirements (e.g. countries might wish to operate with a
exactly equals the endotoxin limit concentration, gelation will lower average body weight of 60 kg instead of 70 kg frequently
occur, as is the case when the endotoxin concentration is much employed in Europe) or any additional safety margins
higher, and the substance or product will fail the test, because requested by the competent authority.
the all-or-none character of the test makes it impossible to Formulation of the product. The limit must take into
differentiate between a concentration exactly equal to the consideration any theoretical bacterial endotoxin load
endotoxin limit concentration and one that is higher. It is only introduced by any other components used for reconstitution
when no gelation occurs that the analyst may conclude that and/or dilution of the product (e.g. water for injections) or
the endotoxin concentration is below the endotoxin limit. introduced by starting materials and/or raw materials.
For substances or products in the solid state, this endotoxin 2-5. MAXIMUM VALID DILUTION
limit concentration per mass unit or per International Unit Which dilution of the substance or product is to be used in the
(IU) of substance or product has to be converted into a test to obtain maximal assurance that a negative result means
concentration of endotoxin per millilitre of solution to be that the endotoxin concentration of the substance or product
examined, as the test can only be carried out on a solution. is less than the endotoxin limit and that a positive result means
The case of substances or products that already exist in the that the lysate detected an endotoxin concentration equal to
liquid state (such as infusion fluids) is discussed below. or greater than the endotoxin limit? This dilution depends on
2-3. CALCULATION OF THE ENDOTOXIN LIMIT the endotoxin limit and on the sensitivity of the lysate ; it is
The endotoxin limit for active substances administered called the maximum valid dilution (MVD) and its value may
parenterally, defined on the basis of dose, is equal to : be calculated using the following expression :

K endotoxin limit × concentration of test solution

λ
M

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

EUROPEAN PHARMACOPOEIA 10.0 5.1.10. Guidelines for using the test for bacterial endotoxins

Concentration of test solution : Production process (chemical synthesis, fermentation,

– in mg/mL if the endotoxin limit is specified by mass biotechnological method). For products of fermentation,
(IU/mg) ; the expression system is to be considered (prokaryotic,
eukaryotic) and, for a prokaryotic expression system, whether
– in Units/mL if the endotoxin limit is specified by unit of
gram-positive or gram-negative bacteria are used. Also, the
biological activity (IU/Unit) ;
culture media components are examined with consideration
– in mL/mL if the endotoxin limit is specified by volume given to their origin (synthetic, animal, plant).
(IU/mL).
Bioburden. The potential presence of gram-positive bacteria
λ = the labelled lysate sensitivity in the gel-clot technique and fungi as contaminants of the active substance, excipients
(IU/mL) or the lowest concentration used in the or starting materials and raw materials used in the production
standard curve of the turbidimetric or chromogenic of the medicinal product, and the origin of the raw materials
techniques. (synthetic, animal, plant) have to be taken into consideration.
When the value of the MVD is not a whole number, a The quality of the water plays an important role on the overall
convenient whole number smaller than the MVD may be evaluation.
used for routine purposes (which means preparing a solution Capability of the downstream process. It must be verified
of the substance or product that is less diluted than the whether bacterial endotoxin removal steps are part of the
MVD indicates). In this case, a negative result indicates that downstream process.
the endotoxin concentration of the substance or product
lies below the limit value. However, when the endotoxin Safety. The target population and the route of administration
concentration of the substance or product in such a test is less (e.g. intravenous, intrathecal) have to be taken into account
than the endotoxin limit but high enough to make the reaction in the risk assessment.
with the lysate result in a clot, the test may be positive under Stability of the detectable endotoxins. It has to be considered
these conditions. Hence, when a test with this ‘convenient’ that the ability to detect endotoxins can be affected by
dilution factor is positive, the substance or product is diluted interaction with certain components, storage conditions or
to the MVD and the test is repeated. In any case of doubt or storage time, temperature and handling of the test sample.
dispute, the MVD must be used. Procedures that demonstrate stability of the detectable
This stresses the importance of the confirmation of the endotoxin content have to be established for storing, handling
sensitivity of the lysate. and mixing of samples.
Example
4. REFERENCE MATERIAL
A 50 mg/mL solution of phenytoin sodium (intended for
intravenous injection) has to be tested. Determine the MVD, Endotoxin standard BRP is intended for use as the reference
given the following variables : preparation. It has been assayed against the WHO
M = maximum human dose = 15 mg per kilogram of International Standard for Endotoxin and its potency is
body mass ; expressed in International Units of endotoxin per vial. The
c = 50 mg/mL ; International Unit of endotoxin is defined as the specific
activity of a defined mass of the International Standard.
K = 5 IU of endotoxin per kilogram of body mass ;
For routine purposes, another preparation of endotoxin may
λ = 0.4 IU of endotoxin per millilitre. be used, provided it has been assayed against the International
Standard for Endotoxin or the BRP and its potency is
5 × 50 1 expressed in International Units of endotoxin.
MVD = × = 41.67
15 0.4 NOTE : 1 International Unit (IU) of endotoxin is equal to
For routine tests on this product, it may be expedient to 1 Endotoxin Unit (E.U.).
dilute 1 mL of the solution to be examined to 20 mL (MVD/2
rounded to the next lower whole number). However, if this 5. WATER FOR BET
test result is positive the analyst will have to dilute 1 mL to Water for BET is sterile water that is free of detectable levels of
41.67 mL and repeat the test. A dilution to 41.67 mL is also endotoxin. Usually it is commercially available and certified.
necessary when the test is performed to settle a dispute.
General chapter 2.6.14. Bacterial endotoxins indicates that
3. RISK ASSESSMENT methods other than triple distillation may be used to prepare
As stated in section 1 of this general chapter, the conclusion water for BET. Reverse osmosis has been used with good
is generally justified that the absence of bacterial endotoxins results ; some analysts may prefer to distil the water more than
in a substance or product implies the absence of pyrogenic 3 times. Whatever method is used, the resultant product must
components, provided the presence of non-endotoxin be free of detectable bacterial endotoxins.
pyrogenic substances can be ruled out. To rule out the
presence of non-endotoxin pyrogens in substances or 6. pH OF THE MIXTURE
products, the use of the monocyte-activation test (2.6.30) In the test for bacterial endotoxins, optimum gel-clot occurs
is recommended at release or during development of for a mixture at pH 6.0-8.0. However, the addition of the lysate
the production process ; if any changes are made to the to the sample may result in a lowering of the pH.
production process that could influence the quality of the
product regarding pyrogenicity, the monocyte-activation 7. VALIDATION OF THE LYSATE
test is repeated. Examples of such changes include the use
of different raw materials, a different production site and It is important to follow the manufacturer’s instructions for
different process parameters. the preparation of the solutions of the lysate.
The decision to use the test for bacterial endotoxins as the sole The positive end-point dilution factors in gel-clot methods A
pyrogenicity test is to be made after careful evaluation of the and B are converted to logarithms. The reason is that if the
risk of the substance or product containing non-endotoxin frequency distribution of these logarithmic values is plotted, it
pyrogens. The risk assessment is made with consideration usually approaches a normal distribution curve much more
given to any factor that could result in the inclusion of closely than the frequency distribution of the dilution factors
pyrogens not detected by the test for bacterial endotoxins. The themselves ; in fact it is so similar that it is acceptable to use
items below constitute a non-exhaustive list of factors to be the normal frequency distribution as a mathematical model
considered in the risk assessment. and to calculate confidence limits with Student’s t-test.

General Notices (1) apply to all monographs and other texts 641
5.1.10. Guidelines for using the test for bacterial endotoxins EUROPEAN PHARMACOPOEIA 10.0

8. PRELIMINARY TEST FOR INTERFERING FACTORS the test (for method A and B). The purpose of the negative
control is to verify the absence of a detectable concentration
Some substances or products cannot be tested directly for the of endotoxin in the water for BET.
presence of bacterial endotoxins because they are not miscible
with the reagents, they cannot be adjusted to pH 6.0-8.0 or they The positive control, which contains the product to be
inhibit or activate enzymatic reaction (such as β-D-glucans). examined at the concentration used in the test, is intended to
show the absence of inhibiting factors at the time and under
Therefore a preliminary test is required to check for the the conditions of the test.
presence of interfering factors ; when these are found the
analyst must demonstrate that the procedure to remove them
has been effective and that by applying this procedure, any 11. READING AND INTERPRETATION OF RESULTS
bacterial endotoxins present have not been removed.
Minute amounts of bacterial endotoxin in the water for BET,
The object of the preliminary test is to test the null hypothesis or in any other reagent or material to which the lysate is
that the sensitivity of the lysate in the presence of the substance exposed during the test, may escape detection as long as they
or product to be examined does not differ significantly from do not reach the sensitivity limit of the lysate. However, they
the sensitivity of the lysate in the absence of the product. may raise the amount of bacterial endotoxin in the solution
A simple criterion is used in methods A and B : the null containing the substance or product to be examined to just
hypothesis is accepted when the sensitivity of the lysate in the above the sensitivity limit and cause a positive reaction.
presence of the product is at least 0.5 times and not more than
twice the sensitivity of the lysate by itself. The risk of this happening may be reduced by testing the
water for BET and the other reagents and materials with the
The test for interfering factors in gel-clot methods A and B most sensitive lysate available, or at least one that is more
requires the use of a sample of the substance or product in sensitive than the one used in the test on the product. Even
which no endotoxins are detectable. This presents a theoretical then, the risk of such a ‘false positive result’ cannot be ruled
problem when an entirely new product has to be tested. out completely.
Hence, a different approach was designed for quantitative
methods C, D, E and F.
12. REPLACEMENT OF METHODS PRESCRIBED IN
Note that methods D and E, which use a chromogenic peptide, MONOGRAPHS
require reagents that are absent in methods A, B, C and F,
and hence compliance of methods A, B, C or F with the 12-1. REPLACEMENT BY ANOTHER PH. EUR. METHOD
requirements for interfering factors cannot be extrapolated to As stated in the General Notices, the test methods given in
method D or method E without further testing. monographs and general chapters have been validated in
accordance with accepted scientific practice and current
recommendations on analytical validation. The methods
9. REMOVAL OF INTERFERING FACTORS described in general chapters 2.6.14. Bacterial endotoxins
and 2.6.30. Monocyte-activation test therefore do not have
The procedures to remove interfering factors must not to be re-validated per se, other than in consideration of their
increase or decrease (for example, by adsorption) the amount use for a specific substance or product in a specific analytical
of endotoxin in the substance or product to be examined. The environment.
correct way of checking this is to apply the procedures to a
spiked sample of the substance or product to be examined, The procedure and the materials and reagents used in the
that is, a sample to which a known amount of endotoxin method must be validated as described for the test concerned.
has been added, and then to measure the recovery of the The absence of interfering factors (and, if necessary, the
endotoxin after the removal process has been conducted. procedure for removing them) is verified on samples of at least
Methods C and D. If the nature of the product to be examined 3 production batches.
results in an interference that cannot be removed by classical
methods (e.g. dilution or centrifugation), it may be possible to The necessary information is sought from manufacturers ;
determine the standard curve in the same type of substance or companies are invited to provide any validation data that they
product freed from endotoxins by appropriate treatment or by have concerning the applicability of the replacement test to
dilution of the substance or product. The endotoxins test is the substances and products of interest ; such data includes
then carried out by comparison with this standard curve. details of sample preparation and of any procedures necessary
to eliminate interfering factors.
Ultrafiltration with cellulose triacetate asymmetric membrane
filters has been found to be suitable in most cases. The filters As stated in general chapter 2.6.30. Monocyte-activation
must be properly validated, because under some circumstances test, the monocyte-activation test is primarily intended as
cellulose derivatives (β-D-glucans) can cause false positive a replacement of the rabbit pyrogen test. Guidelines on
results. which methods to use (A, B or C) and on how to validate
the monocyte-activation test are described in general chapter
Another option to remove interfering factors is a 2-step 2.6.30. Monocyte-activation test.
procedure in which 1) endotoxin within the interfering 12-2. REPLACEMENT BY AN ALTERNATIVE METHOD
sample is fixed on a solid phase, and 2) after removal of the NOT DESCRIBED IN THE PH. EUR.
interfering substance (e.g. by washing) the endotoxin is The use of alternative reagents such as recombinant factor C
detected unimpaired under suitable testing conditions. as a replacement to the amoebocyte lysate eliminates the use
of a reagent extracted from live animals.

10. THE PURPOSE OF THE CONTROLS Replacement of a rabbit pyrogen test or a bacterial endotoxin
test prescribed in a monograph by a test using recombinant
The purpose of the control made up with water for BET factor C reagent or any other reagent as a replacement of the
and the reference preparation of endotoxin at twice the amoebocyte lysate is to be regarded as the use of an alternative
concentration of the labelled lysate sensitivity is to verify the method in the replacement of a pharmacopoeial test, as
activity of the lysate at the time and under the conditions of described in the General Notices.

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

EUROPEAN PHARMACOPOEIA 10.0 5.1.11. Bactericidal, fungicidal or yeasticidal activity of antiseptics

07/2017:50111 Strains for bactericidal activity testing

Staphylococcus aureus such as ATCC 6538, NCIMB 9518,
CIP 4.83, NBRC 13276
Candida albicans such as ATCC 10231, NCPF 3179,
CIP 48.72, NBRC 1594
5.1.11. DETERMINATION OF Yeast growth conditions
BACTERICIDAL, FUNGICIDAL Sabouraud-dextrose agar or number of CFU in test suspension :
OR YEASTICIDAL ACTIVITY OF Sabouraud-dextrose broth 1-5 × 107 CFU/mL

ANTISEPTIC MEDICINAL PRODUCTS - for preparation of test strains :

20-25 °C, 2-3 days and subculture
This general chapter describes a test that can be used for at least twice
the determination of antimicrobial activity in antiseptic - for testing of the product
and validation of the test :
medicinal products that are miscible with water and intended 20-25 °C, ≤ 5 days
for administration by direct contact with the skin or mucous
membranes. The extent of testing is dependent on the declared Strains for fungicidal activity testing
antimicrobial activity of the product. Candida albicans such as ATCC 10231, NCPF 3179,
CIP 48.72, NBRC 1594
The test determines whether a product exhibits bactericidal,
fungicidal or yeasticidal activity and complies with an Aspergillus brasiliensis such as ATCC 16404, IMI 149007,
established specification for such activity. CIP 1431.83, NBRC 9455

This test cannot replace or confirm the assessment of the clinical Fungal growth conditions
efficacy of such preparations. Sabouraud-dextrose agar or number of CFU in test suspension :
Sabouraud-dextrose broth 1-5 × 107 CFU/mL
1. PRINCIPLE - for preparation of test suspension
of C. albicans : 20-25 °C, 2-3 days
Antimicrobial activity is determined by adding test
suspensions of micro-organisms (bacteria, fungi or yeasts, - for preparation of test suspension
of A. brasiliensis spores : 20-25 °C, at
separately) to the sample antiseptic product. The mixture least 5 days until good sporulation
is maintained at 33 ± 1 °C for contact times of 5 min for - for testing of the product
bactericidal activity and 15 min for fungicidal or yeasticidal and validation of the test with
activity. Additional contact times may be chosen, according to C. albicans and A. brasiliensis :
the stated use of the antiseptic medicinal product. At the end 20-25 °C, ≤ 5 days
of the contact time, an aliquot is taken and the antimicrobial The recommended solutions and media are described in
activity in this aliquot is immediately stopped by a validated general chapter 2.6.13. Purified water is used. All reagents
method. 2 methods are available : dilution-neutralisation and are sterile prior to use.
membrane filtration.
The procedure is validated to verify its ability to demonstrate The test for bactericidal, fungicidal or yeasticidal activity
the required reduction in the count of viable micro-organisms is performed with the designated strains as described in
by the use of appropriate controls. Table 5.1.11.-1. In addition to these micro-organisms, it may
be necessary to add other bacterial or fungal strains that
2. TEST MICRO-ORGANISMS AND GROWTH represent the indications of the antiseptic medicinal product
CONDITIONS tested.
Prepare standardised stable suspensions of test strains as Single-strain challenges are used. The counts are performed in
stated in section 2-1. Seed-lot culture maintenance techniques duplicate and the arithmetic mean of the results is calculated
(seed-lot systems) are used so that the viable micro-organisms and expressed in CFU/mL.
used for inoculation are not more than 5 passages removed 2-1. PREPARATION OF TEST SUSPENSION
from the original master seed-lot. Grow each of the microbial For harvesting the micro-organisms use a sufficient volume
test strains separately as described in Table 5.1.11.-1. of a 9 g/L solution of sodium chloride R (for bacteria and
C. albicans) or a solution containing 9 g/L of sodium chloride R
and 0.5 g/L of polysorbate 80 R (for A. brasiliensis), to obtain
Table 5.1.11.-1. – Test micro-organisms and growth conditions a test suspension with the number of CFU described in
Strains for bactericidal activity testing Table 5.1.11.-1. Use the suspension within 2 h or within 24 h
if stored at 2-8 °C.
Staphylococcus aureus such as ATCC 6538, NCIMB 9518,
CIP 4.83, NBRC 13276 2-2. PREPARATION OF ANTISEPTIC PRODUCT TEST
SOLUTION
Enterococcus hirae such as ATCC 10541, NCIMB 8192,
CIP 58.55, DSM 3320 The concentration of the antiseptic product test solution shall
be, if possible, 1.25 times the in-use test concentration because
Escherichia coli such as NCIMB 10083, CIP 54.117,
NCTC 10538, DSM 11250
it is diluted to 80 per cent during the test and the method
validation.
Pseudomonas aeruginosa such as ATCC 15442, NCIMB 8626,
CIP 103467, NBRC 13275 2-3. NEUTRALISING AGENTS
Bacterial growth conditions Neutralising agents are used to neutralise the antimicrobial
activity of the antiseptic product. The common neutralising
Casein soya bean digest agar or number of CFU in test suspension : agents are listed in Table 2.6.12.-2 of general chapter 2.6.12.
casein soya bean digest broth 1-5 × 108 CFU/mL Microbiological examination of non-sterile products : microbial
- for preparation of test strains : enumeration tests. The neutralisation time is not less than 10 s
30-35 °C, 18-24 h and subculture at
least twice and not more than 60 s.
- for testing of the product
and validation of the test : 3. METHODS
30-35 °C, ≤ 3 days
Prior to testing, equilibrate the temperature of all reagents
Strain for yeasticidal activity testing
to 33 ± 1 °C.

General Notices (1) apply to all monographs and other texts 643