EUROPEAN PHARMACOPOEIA 8.0 5.1.1.

Methods of preparation of sterile products

5.1. GENERAL TEXTS ON guaranteed nor can it be demonstrated. The inactivation

MICROBIOLOGY
of micro-organisms by physical or chemical means follows
an exponential law ; thus there is always a nite statistical
probability that a micro-organism may survive the sterilising
01/2008:50101 process. For a given process, the probability of survival
is determined by the number, types and resistance of the
5.1.1. METHODS OF PREPARATION OF micro-organisms present and by the environment in which
the organisms exist during treatment. The SAL of a sterilising
STERILE PRODUCTS process is the degree of assurance with which the process in
Sterility is the absence of viable micro-organisms. The sterility question renders a population of items sterile. The SAL for a
of a product cannot be guaranteed by testing ; it has to be given process is expressed as the probability of a non-sterile
assured by the application of a suitably validated production item in that population. An SAL of 10 6, for example, denotes
process. It is essential that the effect of the chosen sterilisation a probability of not more than one viable micro-organism
procedure on the product (including its nal container or in 1 106 sterilised items of the nal product. The SAL of
package) is investigated to ensure effectiveness and the a process for a given product is established by appropriate
integrity of the product and that the procedure is validated validation studies.
before being applied in practice. It is recommended that METHODS AND CONDITIONS OF STERILISATION
the choice of the container is such as to allow the optimum
Sterilisation may be carried out by one of the methods
sterilisation to be applied. Failure to follow meticulously a
described below. Modications to, or combinations of, these
validated process involves the risk of a non-sterile product
methods may be used provided that the chosen procedure
or of a deteriorated product. Revalidation is carried out
is validated both with respect to its effectiveness and the
whenever major changes in the sterilisation procedure,
integrity of the product including its container or package.
including changes in the load, take place. It is expected that
the principles of good manufacturing practice (as described For all methods of sterilisation the critical conditions of
in, for example, the European Community Guide to GMP) the operation are monitored in order to conrm that the
will have been observed in the design of the process including, previously determined required conditions are achieved
in particular, the use of : throughout the batch during the whole sterilisation process
qualied personnel with appropriate training, This applies in all cases including those where the reference
conditions are used.
adequate premises,
TERMINAL STERILISATION
suitable production equipment, designed for easy cleaning
and sterilisation, For terminal sterilisation it is essential to take into account the
non-uniformity of the physical and, where relevant, chemical
adequate precautions to minimise the bioburden prior to conditions within the sterilising chamber. The location within
sterilisation, the sterilising chamber that is least accessible to the sterilising
validated procedures for all critical production steps, agent is determined for each loading conguration of each
environmental monitoring and in-process testing type and size of container or package (for example, the coolest
procedures. location in an autoclave). The minimum lethality delivered by
The precautions necessary to minimise the pre-sterilisation the sterilising cycle and the reproducibility of the cycle are also
bioburden include the use of components with an acceptable determined in order to ensure that all loads will consistently
low degree of microbial contamination. Microbiological receive the specied treatment.
monitoring and setting of suitable action limits may be Having established a terminal sterilisation process, knowledge
advisable for ingredients which are liable to be contaminated of its performance in routine use is gained wherever possible,
because of their origin, nature or method of preparation. by monitoring and suitably recording the physical and, where
The methods described here apply mainly to the inactivation relevant, chemical conditions achieved within the load in the
or removal of bacteria, yeasts and moulds. For biological chamber throughout each sterilising cycle.
products of animal or human origin or in cases where such Steam sterilisation (Heating in an autoclave). Sterilisation
material has been used in the production process, it is necessary by saturated steam under pressure is preferred, wherever
during validation to demonstrate that the process is capable of applicable, especially for aqueous preparations. For this
the removal or inactivation of relevant viral contamination. method of terminal sterilisation the reference conditions for
Guidance on this aspect is provided in, for example, the aqueous preparations are heating at a minimum of 121 C for
appropriate European Community Notes for Guidance. 15 min. Other combinations of time and temperature may be
Wherever possible, a process in which the product is sterilised used provided that it has been satisfactorily demonstrated that
in its nal container (terminal sterilisation) is chosen. When the process chosen delivers an adequate and reproducible level
a fully validated terminal sterilisation method by steam, dry of lethality when operating routinely within the established
heat or ionising radiation is used, parametric release, that is tolerances. The procedures and precautions employed are
the release of a batch of sterilised items based on process data such, as to give an SAL of 10 6 or better. Guidance concerning
rather than on the basis of submitting a sample of the items to validation by means of the F0 concept is provided below (5.1.5).
sterility testing, may be carried out, subject to the approval of Knowledge of the physical conditions (temperature and
the competent authority. pressure) within the autoclave chamber during the sterilisation
If terminal sterilisation is not possible, ltration through procedure is obtained. The temperature is usually measured
a bacteria-retentative lter or aseptic processing is used ; by means of temperature-sensing elements inserted into
wherever possible, appropriate additional treatment of the representative containers together with additional elements at
product (for example, heating of the product) in its nal the previously established coolest part of the loaded chamber.
container is applied. In all cases, the container and closure are The conditions throughout each cycle are suitably recorded,
required to maintain the sterility of the product throughout for example, as a temperature-time chart, or by any other
its shelf-life. suitable means.
Sterility Assurance Level (SAL) Where a biological assessment is carried out, this is obtained
Where appropriate reference is made within the methods using a suitable biological indicator (5.1.2).
described below, to a sterility assurance level or SAL. The Dry heat sterilisation. For this method of terminal
achievement of sterility within any one item in a population sterilisation the reference conditions are a minimum of 160 C
of items submitted to a sterilisation process cannot be for at least 2 h. Other combinations of time and temperature

General Notices (1) apply to all monographs and other texts 555
5.1.2. Biological indicators of sterilisation
may be used provided that it has been satisfactorily
demonstrated that the process chosen delivers an adequate and
reproducible level of lethality when operated routinely within
the established tolerances. The procedures and precautions
employed are such as to give an SAL of 10 6 or better.
Dry heat sterilisation is carried out in an oven equipped
with forced air circulation or other equipment specially
designed for the purpose. The steriliser is loaded in such
a way that a uniform temperature is achieved throughout
the load. Knowledge of the temperature within the steriliser
during the sterilisation procedure is usually obtained
by means of temperature-sensing elements inserted into
representative containers together with additional elements at
the previously established coolest part of the loaded steriliser.
The temperature throughout each cycle is suitably recorded.
Where a biological assessment is carried out, this is obtained
using a suitable biological indicator (5.1.2).
Dry heat at temperatures greater than 220 C is frequently
used for sterilisation and depyrogenation of glassware. In this
case demonstration of a 3 log10 reduction in heat resistant
endotoxin can be used as a replacement for biological
indicators (5.1.2).
Ionising radiation sterilisation. Sterilisation by this method
is achieved by exposure of the product to ionising radiation
in the form of gamma radiation from a suitable radioisotopic
source (such as cobalt 60) or of a beam of electrons energised
by a suitable electron accelerator.
In some countries there are regulations that lay down rules
for the use of ionising radiation for sterilisation purposes, for
example, in the appropriate European Community Notes for
Guidance.
For this method of terminal sterilisation the reference
absorbed dose is 25 kGy. Other doses may be used provided
that it has satisfactorily been demonstrated that the dose
chosen delivers an adequate and reproducible level of lethality
when the process is operated routinely within the established
tolerances. The procedures and precautions employed are
such as to give an SAL of 10 6 or better.
During the sterilisation procedure the radiation absorbed by
the product is monitored regularly by means of established
dosimetry procedures that are independent of dose rate.
Dosimeters are calibrated against a standard source at a
reference radiation plant on receipt from the supplier and at
suitable intervals of not longer than one year thereafter.
Where a biological assessment is carried out, this is obtained
using a suitable biological indicator (5.1.2).
Gas sterilisation. This method of sterilisation is only to be
used where there is no suitable alternative. It is essential
that penetration by gas and moisture into the material to
be sterilised is ensured and that it is followed by a process
of elimination of the gas under conditions that have been
previously established to ensure that any residue of gas or its
transformation products in the sterilised product is below the
concentration that could give rise to toxic effects during use of
the product. Guidance on this aspect with respect to the use
of ethylene oxide is provided, for example, in the appropriate
European Community Notes for Guidance.
Wherever possible, the gas concentration, relative humidity,
temperature and duration of the process are measured
and recorded. Measurements are made where sterilisation
conditions are least likely to be achieved, as determined at
validation.
The effectiveness of the process applied to each sterilisation
load is checked using a suitable biological indicator (5.1.2).
A suitable sample of each batch is tested for sterility (2.6.1)
before the batch is released.
FILTRATION
Certain active ingredients and products that cannot be
terminally sterilised may be subjected to a ltration procedure
using a lter of a type that has been demonstrated to be
556
EUROPEAN PHARMACOPOEIA 8.0
satisfactory by means of a microbial challenge test using a
suitable test micro-organism. A suspension of Pseudomonas
diminuta (ATCC 19146, NCIMB 11091 or CIP 103020) may
be suitable. It is recommended that a challenge of at least
107 CFU per cm2 of active lter surface is used and that the
suspension is prepared in tryptone soya broth which, after
passage throug the lter, is collected aseptically and incubated
aerobically at 32 C. Such products need special precautions.
The production process and environment are designed to
minimise microbial contamination and are regularly subjected
to appropriate monitoring procedures. The equipment,
containers and closures and, wherever possible, the ingredients
are subjected to an appropriate sterilisation process. It is
recommended that the ltration process is carried out as
close as possible to the lling point. The operations following
ltration are carried out under aseptic conditions.
Solutions are passed through a bacteria-retentive membrane
with a nominal pore size of 0.22 m or less or any other
type of lter known to have equivalent properties of bacteria
retention. Appropriate measures are taken to avoid loss of
solute by adsorption on to the lter and to avoid the release
of contaminants from the lter. Attention is given to the
bioburden prior to ltration, lter capacity, batch size and
duration of ltration. The lter is not used for a longer period
than has been approved by validation of the combination of
the lter and the product in question.
The integrity of an assembled sterilising lter is veried before
use and conrmed after use by carrying out tests appropriate
to the type of lter used and the stage of testing, for example
bubble-point, pressure hold or diffusion rate tests.
Due to the potential additional risks of the ltration method
as compared with other sterilisation processes, a preltration
through a bacteria-retentative lter may be advisable in cases
where a low bioburden cannot be ensured by other means.
ASEPTIC PREPARATION
The objective of aseptic processing is to maintain the sterility
of a product that is assembled from components, each of
which has been sterilised by one of the above methods. This is
achieved by using conditions and facilities designed to prevent
microbial contamination. Aseptic processing may include
aseptic lling of products into container/closure systems,
aseptic blending of formulations followed by aseptic lling
and aseptic packaging.
In order to maintain the sterility of the components and the
product during processing, careful attention needs to be given
to :
environment,
personnel,
critical surfaces,
container/closure sterilisation and transfer procedures,
maximum holding period of the product before lling into
the nal container.
Process validation includes appropriate checks on all the
above and checks on the process are regularly carried out by
means of process simulation tests using microbial growth
media which are then incubated and examined for microbial
contamination (media ll tests). In addition, a suitable sample
of each batch of any product that is sterilised by ltration
and/or aseptically processed is tested for sterility (2.6.1) before
the batch is released.
01/2011:50102
5.1.2. BIOLOGICAL INDICATORS OF
STERILISATION
Biological indicators are standardised preparations of
selected micro-organisms used to assess the effectiveness
of a sterilisation procedure. They usually consist of a
population of bacterial spores placed on a suitable inert
See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 8.0
carrier. The inoculated carrier is covered in such a way that it
is protected from any deterioration or contamination, while
allowing the sterilising agent to enter into contact with the
micro-organisms. Spore suspensions may be presented in
sealed ampoules. Biological indicators are prepared in such
a way that they can be stored under dened conditions ; an
expiry date is set.
Micro-organisms of the same bacterial species as the bacteria
used to manufacture the biological indicators may be
inoculated directly into a liquid product to be sterilised or
into a liquid product similar to that to be sterilised. In this
case, it must be demonstrated that the liquid product has no
inhibiting effect on the spores used, especially as regards their
germination.
A biological indicator is characterised by the name of the
species of bacterium used as the reference micro-organism,
the number of the strain in the original collection, the number
of viable spores per carrier and the D-value. The D-value is
the value of a parameter of sterilisation (duration or absorbed
dose) required to reduce the number of viable organisms to
10 per cent of the original number. It is of signicance only
under precisely dened experimental conditions. Only the
stated micro-organisms are present. Biological indicators
consisting of more than one species of bacteria on the same
carrier may be used. Information on the culture medium and
the incubation conditions is supplied.
It is recommended that the indicator organisms are placed
at the locations presumed, or wherever possible, found by
previous physical measurement to be least accessible to the
sterilising agent. After exposure to the sterilising agent,
aseptic technique is used to transfer carriers of spores to the
culture media, so that no contamination is present at the time
of examination. Biological indicators that include an ampoule
of culture medium placed directly in the packaging protecting
the inoculated carrier may be used.
A choice of indicator organisms is made such that :
a) the resistance of the test strain is suitable for the particular
sterilisation method and is great compared to the resistance of
micro-organisms potentially contaminating the product ;
b) the test strain is non-pathogenic ;
c) the test strain is easy to culture.
After incubation, growth of the reference micro-organisms
subjected to a sterilisation procedure indicates that the
procedure has been unsatisfactory.
Steam sterilisation. The use of biological indicators intended
for steam sterilisation is recommended for the validation of
sterilisation cycles. Spores of Geobacillus stearothermophilus
(for example, ATCC 7953, NCTC 10007, NCIMB 8157
or CIP 52.81) or other strains of micro-organisms having
demonstrated equivalent performance are recommended.
The number of viable spores exceeds 5 105 per carrier. The
D-value at 121 C is not less than 1.5 min. It is veried that
exposing the biological indicators to steam at 121 1 C for
6 min leaves revivable spores, and that there is no growth of
the reference micro-organisms after the biological indicators
have been exposed to steam at 121 1 C for 15 min.
Dry-heat sterilisation. Spores of Bacillus atrophaeus (for
example, ATCC 9372, NCIMB 8058 or CIP 77.18) or other
strains of micro-organisms having demonstrated equivalent
performance are recommended for the preparation of
biological indicators. The number of viable spores exceeds
1 106 per carrier and the D-value at 160 C is not less
than 2.5 min. Dry heat at temperatures greater than 220 C
is frequently used for sterilisation and depyrogenation of
glassware. In this case, demonstration of a 3 log10 reduction in
heat-resistant bacterial endotoxin can be used as a replacement
for biological indicators.
Ionising radiation sterilisation. Biological indicators may
be used to monitor routine operations, as an additional
possibility to assess the effectiveness of the set dose of
General Notices (1) apply to all monographs and other texts
5.1.3. Efcacy of antimicrobial preservation
radiation energy, especially in the case of accelerated electron
sterilisation. The spores of Bacillus pumilus (for example,
ATCC 27142, NCTC 10327, NCIMB 10692 or CIP 77.25)
or other strains of micro-organisms having demonstrated
equivalent performance are recommended. The number of
viable spores exceeds 1 107 per carrier. The D-value is not
less than 1.9 kGy. It is veried that there is no growth of the
reference micro-organisms after the biological indicators have
been exposed to 25 kGy (minimum absorbed dose).
Gas sterilisation. The use of biological indicators is necessary
for all gas sterilisation procedures, both for the validation
of the cycles and for routine operations. Gas sterilisation
is widely used for medical devices, isolators, chambers, etc.
Use for such purposes is outside the scope of the European
Pharmacopoeia. The use of spores of Bacillus atrophaeus (for
example, ATCC 9372, NCIMB 8058 or CIP 77.18) or other
strains of micro-organisms having demonstrated equivalent
performance is recommended for ethylene oxide. The number
of viable spores exceeds 1 106 per carrier. The parameters
of resistance are the following : the D-value is not less than
2.5 min for a test cycle involving 600 mg/L of ethylene oxide,
at 54 C and at 60 per cent relative humidity. It is veried that
there is no growth of the reference micro-organisms after
the biological indicators have been exposed to the test cycle
described above for 25 min and that exposing the indicators
to a reduced temperature cycle (600 mg/L, 30 C and 60 per
cent relative humidity) for 50 min leaves revivable spores.
01/2011:50103
5.1.3. EFFICACY OF ANTIMICROBIAL
PRESERVATION
If a pharmaceutical preparation does not itself have adequate
antimicrobial activity, antimicrobial preservatives may be
added, particularly to aqueous preparations, to prevent
proliferation or to limit microbial contamination which,
during normal conditions of storage and use, particularly for
multidose containers, could occur in a product and present
a hazard to the patient from infection and spoilage of the
preparation. Antimicrobial preservatives must not be used as
a substitute for good manufacturing practice.
The efcacy of an antimicrobial preservative may be enhanced
or diminished by the active constituent of the preparation
or by the formulation in which it is incorporated or by the
container and closure used. The antimicrobial activity of the
preparation in its nal container is investigated over the period
of validity to ensure that such activity has not been impaired
by storage. Such investigations may be carried out on samples
removed from the nal container immediately prior to testing.
During development of a pharmaceutical preparation, it
shall be demonstrated that the antimicrobial activity of the
preparation as such or, if necessary, with the addition of
a suitable preservative or preservatives provides adequate
protection from adverse effects that may arise from microbial
contamination or proliferation during storage and use of the
preparation.
The efcacy of the antimicrobial activity may be demonstrated
by the test described below. The test is not intended to be used
for routine control purposes.
TEST FOR EFFICACY OF ANTIMICROBIAL
PRESERVATION
The test consists of challenging the preparation, wherever
possible in its nal container, with a prescribed inoculum of
suitable micro-organisms, storing the inoculated preparation
at a prescribed temperature, withdrawing samples from the
container at specied intervals of time and counting the
organisms in the samples so removed.
557
5.1.3. Efcacy of antimicrobial preservation EUROPEAN PHARMACOPOEIA 8.0

The preservative properties of the preparation are adequate if,
in the conditions of the test, there is a signicant fall or no
increase, as appropriate, in the number of micro-organisms
in the inoculated preparation after the times and at the
temperatures prescribed. The acceptance criteria, in terms of
decrease in the number of micro-organisms with time, vary
for different types of preparations according to the degree of
protection intended (see Tables 5.1.3.-1/2/3).
Test micro-organisms
Pseudomonas aeruginosa ATCC 9027 ; NCIMB 8626 ; CIP 82.118.
ltration (2.6.12). Ensure that any residual antimicrobial
activity of the product is eliminated by dilution, by ltration or
by the use of a specic inactivator. When dilution procedures
are used, due allowance is made for the reduced sensitivity
in the recovery of small numbers of viable micro-organisms.
When a specic inactivator is used, the ability of the system to
support the growth of the test organisms is conrmed by the
use of appropriate controls.
The procedure is validated to verify its ability to demonstrate
the required reduction in count of viable micro-organisms.

Staphylococcus aureus ATCC 6538 ; NCTC 10788 ;

NCIMB 9518 ; CIP 4.83. ACCEPTANCE CRITERIA
Candida albicans ATCC 10231 ; NCPF 3179 ; IP 48.72. The criteria for evaluation of antimicrobial activity are given
Aspergillus brasiliensis ATCC 16404 ; IMI 149007 ; IP 1431.83. in Tables 5.1.3.-1/2/3 in terms of the log10 reduction in the
number of viable micro-organisms against the value obtained
for the inoculum.
Single-strain challenges are used and the designated
micro-organisms are supplemented, where appropriate, by Table 5.1.3.-1. - Parenteral preparations, eye preparations,
other strains or species that may represent likely contaminants intrauterine preparations and intramammary preparations
to the preparation. It is recommended, for example, that
Escherichia coli (ATCC 8739 ; NCIMB 8545 ; CIP 53.126) is Log10 reduction
used for all oral preparations and Zygosaccharomyces rouxii
6h 24 h 7d 14 d 28 d
(NCYC 381 ; IP 2021.92) for oral preparations containing a
high concentration of sugar. Bacteria A 2 3 - - NR
Preparation of inoculum B - 1 3 - NI

Preparatory to the test, inoculate the surface of casein soya Fungi A - 2 - NI -

bean digest agar (2.6.12) for bacteria or Sabouraud-dextrose B - - - 1 NI
agar without the addition of antibiotics (2.6.12) for fungi,
with the recently grown stock culture of each of the specied NR : no recovery.
micro-organisms. Incubate the bacterial cultures at 30-35 C NI : no increase in number of viable micro-organisms compared to
for 18-24 h, the culture of C. albicans at 20-25 C for 48 h, and the previous reading.
the culture of A. brasiliensis at 20-25 C for 1 week or until
good sporulation is obtained. Subcultures may be needed after The A criteria express the recommended efcacy to be
revival before the micro-organism is in its optimal state, but it achieved. In justied cases where the A criteria cannot be
is recommended that their number be kept to a minimum. attained, for example for reasons of an increased risk of
adverse reactions, the B criteria must be satised.
To harvest the bacterial and C. albicans cultures, use a sterile
suspending uid, containing 9 g/L of sodium chloride R, for Table 5.1.3.-2. - Ear preparations, nasal preparations,
dispersal and transfer of the surface growth into a suitable preparations for cutaneous application and preparations for
vessel. Add sufcient suspending uid to reduce the microbial inhalation
count to about 108 micro-organisms per millilitre. To
Log10 reduction
harvest the A. brasiliensis culture, use a sterile suspending
uid containing 9 g/L of sodium chloride R and 0.5 g/L of 2d 7d 14 d 28 d
polysorbate 80 R and adjust the spore count to about 108 per -
Bacteria A 2 3 NI
millilitre by adding the same solution.
B - - 3 NI
Remove immediately a suitable sample from each suspension
and determine the number of colony-forming units per Fungi A - - 2 NI
millilitre in each suspension by plate count or membrane B - - 1 NI
ltration (2.6.12). This value serves to determine the inoculum
and the baseline to use in the test. The suspensions shall be NI : no increase in number of viable micro-organisms compared to
used immediately. the previous reading.

The A criteria express the recommended efcacy to be

METHOD achieved. In justied cases where the A criteria cannot be
attained, for example for reasons of an increased risk of
To count the viable micro-organisms in the inoculated adverse reactions, the B criteria must be satised.
products, use the agar medium used for the initial cultivation
of the respective micro-organisms. Table 5.1.3.-3. - Oral preparations, oromucosal preparations
and rectal preparations
Inoculate a series of containers of the product to be examined,
each with a suspension of one of the test organisms to give Log10 reduction
an inoculum of 105 to 106 micro-organisms per millilitre or
14 d 28 d
per gram of the preparation. The volume of the suspension
of inoculum does not exceed 1 per cent of the volume of the Bacteria 3 NI
product. Mix thoroughly to ensure homogeneous distribution. 1 NI
Fungi
Maintain the inoculated product at 20-25 C, protected from NI : no increase in number of viable micro-organisms compared to
light. Remove a suitable sample from each container, typically the previous reading.
1 mL or 1 g, at zero hour and at appropriate intervals according
to the type of the product and determine the number The above criteria express the recommended efcacy to be
of viable micro-organisms by plate count or membrane achieved.

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

EUROPEAN PHARMACOPOEIA 8.0 5.1.4. Microbiological quality of non-sterile products for pharmaceutical use

01/2014:50104 If it has been shown that none of the prescribed tests will
allow valid enumeration of micro-organisms at the level
5.1.4. MICROBIOLOGICAL QUALITY prescribed, a validated method with a limit of detection as
OF NON-STERILE PHARMACEUTICAL close as possible to the indicated acceptance criterion is used.
In addition to the micro-organisms listed in Table 5.1.4.-1, the
PREPARATIONS AND SUBSTANCES signicance of other micro-organisms recovered is evaluated
FOR PHARMACEUTICAL USE(1) in terms of :
The presence of certain micro-organisms in non-sterile use of the product : hazard varies according to the route of
preparations may have the potential to reduce or even administration (eye, nose, respiratory tract) ;
inactivate the therapeutic activity of the product and has nature of the product : its ability to support growth, the
a potential to adversely affect the health of the patient. presence of adequate antimicrobial preservation ;
Manufacturers therefore have to ensure a low bioburden of method of application ;
nished dosage forms by implementing current guidelines
intended recipient : risk may differ for neonates, infants,
on Good Manufacturing Practice during the manufacture,
the debilitated ;
storage and distribution of pharmaceutical preparations.
Microbial examination of non-sterile products is performed use of immunosuppressive agents, corticosteroids ;
according to the methods given in general chapters 2.6.12 and presence of disease, wounds, organ damage.
2.6.13. Acceptance criteria for non-sterile pharmaceutical Where warranted, a risk-based assessment of the relevant
products based upon the total aerobic microbial count factors is conducted by personnel with specialised training in
(TAMC) and the total combined yeasts/moulds count (TYMC) microbiology and the interpretation of microbiological data.
are given in Tables 5.1.4.-1 and 5.1.4.-2. Acceptance criteria For raw materials, the assessment takes account of processing
are based on individual results or on the average of replicate to which the product is subjected, the current technology of
counts when replicate counts are performed (e.g. direct testing and the availability of materials of the desired quality.
plating methods).
When an acceptance criterion for microbiological quality is Table 5.1.4.-2. Acceptance criteria for microbiological quality
prescribed it is interpreted as follows: of non-sterile substances for pharmaceutical use
1
10 CFU : maximum acceptable count = 20 ; TAMC TYMC
102 CFU : maximum acceptable count = 200 ; (CFU/g or CFU/mL) (CFU/g or CFU/mL)
103 CFU : maximum acceptable count = 2000, and so forth.
Substances for 103 102
Table 5.1.4.-1 includes a list of specied micro-organisms for pharmaceutical use
which acceptance criteria are set. The list is not necessarily
exhaustive and for a given preparation it may be necessary to Recommended acceptance criteria for microbiological quality
test for other micro-organisms depending on the nature of the of herbal medicinal products for oral use and extracts used in
starting materials and the manufacturing process. their preparation are given in general chapter 5.1.8.

Table 5.1.4.-1. Acceptance criteria for microbiological quality of non-sterile dosage forms
TAMC TYMC
Route of administration (CFU/g or (CFU/g or Specied micro-organisms
CFU/mL) CFU/mL)
Non-aqueous preparations for oral use 103 102 Absence of Escherichia coli (1 g or 1 mL)

Aqueous preparations for oral use 10 2

10 1
Absence of Escherichia coli (1 g or 1 mL)

Rectal use 103 102 -

Oromucosal use
Gingival use
Cutaneous use
Nasal use
Auricular use
Vaginal use
Transdermal patches (limits for one patch
including adhesive layer and backing)
Inhalation use (special requirements apply to
liquid preparations for nebulisation)
Special Ph. Eur. provision for oral dosage
forms containing raw materials of natural
(animal, vegetal or mineral) origin for which
antimicrobial pretreatment is not feasible and
for which the competent authority accepts
TAMC of the raw material exceeding 103 CFU/g
or CFU/mL.
Special Ph. Eur. provision for premixes for
medicated feeding stuffs for veterinary use
using excipients of plant origin for which
antimicrobial treatment is not feasible.
Absence of Staphylococcus aureus (1 g or 1 mL)
102 101
Absence of Pseudomonas aeruginosa (1 g or 1 mL)
Absence of Pseudomonas aeruginosa (1 g or 1 mL)
102 101 Absence of Staphylococcus aureus (1 g or 1 mL)
Absence of Candida albicans (1 g or 1 mL)
Absence of Staphylococcus aureus (1 patch)
102 101
Absence of Pseudomonas aeruginosa (1 patch)
Absence of Staphylococcus aureus (1 g or 1 mL)
Absence of Pseudomonas aeruginosa (1 g or 1 mL)
102 101
Absence of bile-tolerant gram-negative
bacteria (1 g or 1 mL)
Not more than 102 CFU of bile-tolerant gram-negative
bacteria (1 g or 1 mL)
104 102 Absence of Salmonella (10 g or 10 mL)
Absence of Escherichia coli (1 g or 1 mL)
Absence of Staphylococcus aureus (1 g or 1 mL)
Not more than 104 CFU of bile-tolerant gram-negative
5 4
bacteria (1 g or 1 mL)
10 10
Absence of Escherichia coli (1 g or 1 mL)
Absence of Salmonella (25 g or 25 mL)

(1) This chapter has undergone pharmacopoeial harmonisation. See chapter 5.8. Pharmacopoeial harmonisation.

General Notices (1) apply to all monographs and other texts 559
5.1.5. Application of the F0 concept to steam sterilisation EUROPEAN PHARMACOPOEIA 8.0

01/2009:50105 assurance for the quality of pharmaceutical products. These

alternative methods may also nd a place in environmental
5.1.5. APPLICATION OF 0 THE F monitoring.
CONCEPT TO STEAM STERILISATION The microbiological methods described in the European
OF AQUEOUS PREPARATIONS Pharmacopoeia have been used for almost a century and these
methods - for enumerating and identifying micro-organisms
The following chapter is published for information. - still serve microbiologists well. Over the years, these
The F0 value of a saturated steam sterilisation process is methods have been invaluable to help control and secure the
the lethality expressed in terms of the equivalent time production of microbiologically-safe pharmaceutical products.
in minutes at a temperature of 121 C delivered by the Nevertheless conventional microbiological methods are slow,
process to the product in its nal container with reference to and results are not available before an incubation period of
micro-organisms possessing a theoretical Z-value of 10. typically up to 14 days. Thus the results from the conventional
microbiological methods seldom enable proactive, corrective
The total F0 of a process takes account of the heating up action to be taken.
and cooling down phases of the cycle and can be calculated
by integration of lethal rates with respect to time at discrete Alternative methods for control of microbiological quality
temperature intervals. have been introduced in recent years, and some of these
When a steam sterilisation cycle is chosen on the basis of the methods have shown potential for real-time or near-real-time
F0 concept, great care must be taken to ensure that an adequate results with the possibility of earlier corrective action. These
assurance of sterility is consistently achieved. In addition to new methods can also offer signicant improvements in the
validating the process, it may also be necessary to perform quality of testing.
continuous, rigorous microbiological monitoring during In this informational chapter new microbiological methods
routine production to demonstrate that the microbiological offering pharmaceutical applications are described. For each
parameters are within the established tolerances so as to give method the basic principle is stated and the benets and
an SAL of 10 6 or better. disadvantages of the method are then discussed. Potential
In connection with sterilisation by steam, the Z-value uses describe applications that may be envisaged in view of the
relates the heat resistance of a micro-organism to changes principles on which the method is based : it is not intended
in temperature. The Z-value is the change in temperature to suggest that actual application has been made. Finally,
required to alter the D-value by a factor of 10. general considerations for the validation of the method are
The D-value (or decimal reduction value) is the value of outlined. These are illustrated by specic examples for each
a parameter of sterilisation (duration or absorbed dose) type of method. A detailed validation protocol is given for
required to reduce the number of viable organisms to 10 per information at the end of this chapter.
cent of the original number. It is only of signicance under
precisely dened experimental conditions. It is not the intention of this chapter to recommend one method
over another, nor is it the intention to provide an exclusive
The following mathematical relationships apply : or exhaustive list of alternative methods that can be used for
pharmaceutical microbiological control. This informational
chapter however may be used in the process of choosing an
D121 = D-value of the reference spores (5.1.2) at 121 C ; alternative microbiological method as a supplement or as an
alternative to conventional microbiological approaches and to
N0 = initial number of viable micro-organisms ; give guidance in the process of validating the chosen method.
N = nal number of viable micro-organisms ; In this rapidly developing eld, further methods are likely to
appear. The guidance offered in this chapter may be equally
IF = inactivation factor. applicable to these methods.
There are 3 major types of determinations specic to
microbiological tests. These include :
qualitative tests for the presence or absence of
D1 = D-value of the micro-organism at temperature T1 ; micro-organisms ;
D2 = D-value of the micro-organism at temperature T2. quantitative tests for enumeration of micro-organisms ;
identication tests.
1-1. QUALITATIVE TESTS FOR THE PRESENCE OR
ABSENCE OF MICRO-ORGANISMS
t = exposure time ; In conventional microbiological analysis this type of test is
D = D-value of micro-organism in the exposure characterised by the use of turbidity or other growth-related
conditions. changes in a culture medium as evidence of the presence of
viable micro-organisms in the test sample. The most common
example of this test is the sterility test. Other examples of this
01/2008:50106 type of testing are those tests designed to evaluate the presence
or absence of a particular type of viable micro-organism in
5.1.6. ALTERNATIVE METHODS FOR a sample.
CONTROL OF MICROBIOLOGICAL 1-2. QUANTITATIVE TESTS FOR ENUMERATION OF
MICRO-ORGANISMS
QUALITY Membrane ltration and plate count methods are
The following chapter is published for information. conventional methods used to estimate the number of viable
micro-organisms present in a sample. The Most Probable
1. GENERAL INTRODUCTION Number (MPN) method is another example of these methods.
The objective of this chapter is to facilitate the implementation MPN was developed as a means to estimate the number of
and use of alternative microbiological methods where this can viable micro-organisms present in a sample not amenable to
lead to cost-effective microbiological control and improved direct plating.

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

EUROPEAN PHARMACOPOEIA 8.0 5.1.6. Alternative methods for control of microbiological quality
1-3. IDENTIFICATION TESTS
Biochemical and morphological characterisation of an
unknown micro-organism is the classical method of
identication used in pharmacopoeial tests. Recently
developed methods have streamlined and automated aspects
of this identication, especially in the areas of data handling,
analysis, and storage. Several new approaches that have been
integrated into these methods include biochemical reactions,
carbon substrate utilisation, characterisation of fatty acid
composition, restriction endonuclease banding patterns and
use of 16S rDNA sequence analysis.
2. GENERAL PRINCIPLES OF ALTERNATIVE METHODS
Alternative microbiological methods employ direct and
indirect methods of detection ; in some instances amplication
of the signal is achieved by enrichment methods. In
recognition of these differences, and for convenience
within this chapter, alternative methods for the control of
microbiological quality are divided into 3 categories :
growth-based methods, where a detectable signal is usually
achieved by a period of subculture ;
direct measurement, where individual cells are
differentiated and visualised ;
cell component analysis, where the expression of specic
cell components offers an indirect measure of microbial
presence.
In some instances, these distinctions are articial but they do
enable a working classication to be created.
2-1. GROWTH-BASED METHODS
2-1-1. Early detection of growth
2-1-1-1. General critical aspects of methods based on early
detection of growth
Such methods are critically dependent upon microbial growth
in order to achieve a detectable number of micro-organisms.
For the typically low levels of microbial contamination seen
in pharmaceutical products, detection may take 24 h or even
more, especially in the case of yeasts and moulds. Increased
sensitivity can be achieved with ltered products. In this case,
after ltration, the membrane is incubated in the medium
and the result is expressed as presence or absence in the
quantity corresponding to the ltered volume. These systems,
because they use an incubation step in liquid media, do
not offer quantitative information but a presence/absence
determination in the quantity analysed. Analysis of more than
one sample quantity may offer a semi-quantitative estimation
(limit test). The major benet of such methods compared to
classical methods frequently resides in the capacity to process
simultaneously a large number of samples and potentially to
obtain a result in a shorter time.
2-1-1-2. Electrochemical methods
Principles of measurement. Micro-organisms multiplying and
metabolising in appropriate growth media produce highly
charged ionic metabolites from weakly charged organic
nutrients leading to the modication of electrical properties
in those media. These changes in impedance (measured by
conductance or capacitance) are monitored with electrodes
included in the culture vessels and in contact with the culture
medium. The measurable end-point is the time taken to detect
a pre-determined impedance change ; the detection time is
inversely proportional to the initial inoculum size. For yeasts
and moulds, which produce only small changes in electrical
impedance, an indirect measurement of conductance using
a potassium hydroxide reservoir is commonly used. Direct
measurement of capacitance can also be carried out.
Critical aspects. Automated detection with electronic data
generation, mapping of the variation of impedance reecting
the growth curve of the micro-organisms, and reduction of
the duration of the test to 48 h.
General Notices (1) apply to all monographs and other texts
Potential uses. Microbiological assay of antibiotics, efcacy
of antimicrobial preservation and presence/absence in the
quantity of sample tested when performing total viable aerobic
count.
2-1-1-3. Measurement of consumption or production of gas
Principles of measurement. Actively multiplying and
metabolising organisms utilise appropriate growth media,
leading to the production of metabolites or elimination
of specic nutrients. In one approach, changes in gaseous
head-space composition may be monitored in closed culture
vessels by pressure transducers responding to gas production
(e.g. CO2) or gas consumption (e.g. O2). Other indicators may
be employed including colorimetric detection of CO2.
Critical aspects. For slow-growing micro-organisms such as
mycobacteria, the method offers more rapid detection. There
is no direct relationship between original microbial burden
and detectable end-point. The incubation temperature and the
algorithm for data processing signicantly affect the results.
Potential uses. Products where slow-growing micro-organisms
may be present.
2-1-1-4. Bioluminescence
Principles of measurement. Adenosine triphosphate (ATP) is
a well-documented marker of cell viability. In this method,
ATP needs rst to be released from micro-organisms using
an appropriate extractant, followed by quantitative assay
using the luciferin/luciferase enzyme system, which emits
light in proportion to the ATP present. The emitted light is
measured with a bioluminometer and is expressed in relative
light units (RLU) for bioluminescence in liquid media. The
RLU obtained from the sample is compared with a threshold
value determined at 2 or 3 times the RLU of the medium used
for cultivation or sample suspension. The result is positive
if the RLU obtained with the analysed sample exceeds the
threshold value. A modication to the method using growth
of micro-organisms captured on a membrane by incubation
on agar medium employs a charge coupled device (CCD)
camera to detect the micro-colonies, and results are expressed
as microCFU. This method is quantitative but has a narrow
range of linearity.
Critical aspects. If the product sampled has a high level of
bacterial contamination (about 500-1000 CFU per sample
quantity tested), the detection is rapid (1 h). For low
levels of contamination (less than 100 CFU per quantity
of sample tested), it is necessary to increase the number of
micro-organisms by an incubation step in culture media
(liquid or solid agar) for 12-48 h according to the method
employed. After this time, in liquid media, one single cell
capable of growth will increase from 1 to 1000 and will be
detected. The yield of ATP varies from one micro-organism to
another, bacteria containing 1-10 fg per cell and fungi around
100 fg per cell and many other factors including the species,
the growth phase of the cell, the nutritional status, the cellular
stress or the cellular age could affect the ATP content of the
cell. Therefore, it is not possible to obtain a count directly
from the RLU value. In addition, turbidity and sample colour
can affect the reaction by either enhancing the reaction and
increasing the level of light output or acting as a quenching
agent and lowering the level of light output. Since the reaction
is enzymatically based, products which could inhibit or
decrease the enzyme activity may interfere. In practice, such
interference is rare but must be thoroughly investigated
during the validation process. The reaction is also sensitive to
the presence of phosphate nucleotides such as ADP or GTP,
which interfere by producing ATP in the presence of adenylate
kinase. This enzyme is used to increase the sensitivity of
some bioluminescence methods : here a 3rd reagent is added
containing ADP and new ATP is produced in the presence of
adenylate kinase released from micro-organisms.
Potential uses. Testing for efcacy of antimicrobial
preservation, presence/absence in the quantity of sample
tested when performing total viable aerobic count
561
5.1.6. Alternative methods for control of microbiological quality
(bioluminescence in tube or microtitre plate), total
viable aerobic count (bioluminescence on membrane),
environmental and water monitoring. The method nds
applications in lterable and non-lterable products.
2-1-1-5. Microcalorimetry
Principles of measurement. Microbial catabolism generates
heat which can be accurately measured by microcalorimetry.
Heat production can be detected by placing the contaminated
sample in a sealed ampoule containing a growth medium
and enclosing within a calorimeter. Using sensitive
instrumentation microbial growth curves can be established.
High bioburdens may be detectable by ow calorimetry.
Critical aspects. Theoretically, this method does not require
microbial growth but simply catabolic activity. Nevertheless,
a minimum number of micro-organisms are required to give
heat output measures above base-line and this is usually
achieved by use of an enrichment method.
Potential uses. Test for efcacy of antimicrobial preservation.
2-1-1-6. Turbidimetry
Principles of measurement. Microbial growth will lead to
detectable changes in medium opacity. This can be accurately
quantied by optical density measurement at a specied
wavelength. In its simplest form such measurements are
performed in a standard spectrophotometer over a wavelength
range generally of 420-615 nm. Alternative automated systems
employ microtitre plate readers offering continuous readout
with early detection of optical density change.
Critical aspects. Attempts have been made to extrapolate the
initial bioburden from the time for detection but this may be
limited to healthy micro-organisms with reproducible growth
characteristics. The methods cannot distinguish between
viable and non-viable micro-organisms.
Potential uses. By means of calibration graphs, determination
of the inoculum size of microbial suspensions for use in
pharmacopoeial tests. In automated mode, establishment of
the preservative sensitivity of test micro-organisms recovered
from formulated products.
2-1-1-7. Phage-based methods
Principles of measurement. Bacterial viruses (bacteriophage,
phage) can infect host cells causing either lysis or incorporation
of their genetic material and expression of novel proteins.
Their high level of host specicity can be employed in
detection methods which exploit the consequences of infection
as an end-point. Such end-points include : plaque formation
on a solid lawn of reporter bacteria ; detection of intracellular
contents released from lysed bacteria (possibly by colorimetric
method) ; or phage-induced effects such as ice nucleation or
bioluminescence following infection by genetically modied
phage. Fluorescently labelled coliphages can be used for the
selective detection of viable E. coli in combination with DEFT
(see 2-3-3.).
Critical aspects. Phage-based detection can be used in both
single and mixed cultures where host specicity allows
both detection and identication. Detectable end-points
often require a minimum number of target cells to ensure a
measurable signal, necessitating enrichment in situations of
low bioburden. The viral infection process can be adversely
affected by sample composition. In most cases there is a
narrow host range which makes it difcult to detect a broad
spectrum of microbial contaminants.
Potential uses. These methods are used mainly for research
purposes with commercial development aimed principally
towards uses in clinical and food microbiology. These
methods are likely to be employed for presence/absence
determinations of specied micro-organisms.
2-1-2. Media development to improve detection
Principles of measurement. Culture media have existed for
many years and have been constantly improved. A recent
innovation is the appearance of chromogenic substrates which
are increasingly used in clinical and food microbiology. The
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EUROPEAN PHARMACOPOEIA 8.0
ability to detect the presence of specic enzymes using suitable
substrates has led to the development of a large number of
methods for the identication of micro-organisms employing
manual or automated methods. The incorporation of such
substrates into a selective or non-selective primary isolation
medium can eliminate the need for further subculture
and biochemical tests to identify certain micro-organisms.
Consequently, chromogenic liquid or solid culture media are
designed to produce specic enzymatic activities for detection
and differentiation of micro-organisms. In these particular
media, dened substrates are introduced into the formulation
and are hydrolysed by the specic cell enzyme of a given
bacteria or fungi during growth. These substrates are chosen
according to the diagnostic enzymatic activity sought and are
linked to coloured indicators.
Critical aspects. The use of innovative media presents several
advantages : improved discrimination of colonies in mixed
culture, ease of use and ease of interpretation. In addition,
response times are shorter because growth and identication
of the micro-organism are simultaneous. However, validation
of the media must be undertaken carefully to ensure a
combination of specicity, selectivity and robustness. The
quality of the signal is based not only on the careful choice
of the enzymes used as the basis of detection, as these
enzymes may be present in different genera, but also on
physico-chemical characteristics of the medium such as pH.
Potential uses. Detection of specied micro-organisms such as
E. coli, coliforms, Salmonella, Staphylococcus and Streptococcus
spp. ; particular benet may be found in presence/absence
testing. Yeasts can also be detected using chromogenic culture
media.
2-2. DIRECT MEASUREMENT
2-2-1. Solid phase cytometry
Principles of measurement. A membrane lter is used to
retain microbial contaminants. Micro-organisms are stained
by labelling using a uorophore as a viability indicator,
either before or after ltration. The uorophore is initially
a non-uorogenic, conjugated substrate that requires
intracellular enzymatic activity to cleave the substrate and
release the uorescent moiety. An intact cellular membrane
is required to retain uorophore within the cytoplasm. Laser
excitation and automated scanning allows the detection of
single, viable uorescent micro-organisms. Appropriate
software permits differentiation of viable micro-organisms
from auto-uorescent particles. The high sensitivity and
rapidity of detection permits near-real-time detection of
microbial contaminants. Total cell counts can be obtained
using a permanently uorescing stain.
Critical aspects. Metabolically active, fastidious and viable
non-culturable micro-organisms can be detected. This may
result in reappraisal of the microbial limits established for
the samples under evaluation. Spores require initiation of
germination to enable detection. Single cell detection may
be achievable, but identication is not currently part of the
routine test protocol. The use of uorescent antibody may
offer a route to selective detection. False positives may occur
from auto-uorescent particles, which can be difcult to
differentiate from micro-organisms.
Potential uses. Rapid and sensitive method for the non-specic
evaluation of bioburden. It has found applications in testing
pharmaceutical-grade waters.
2-2-2. Flow cytometry
Principles of measurement. Fluorophore-labelled
micro-organisms can be detected in suspension as they pass
through a ow cell cytometer. Where a viability-indicating
uorophore substrate is employed, viable micro-organisms
can be differentiated from non-viable particles (see 2-2-1.).
Critical aspects. Flow cytometry may be applied for the
microbiological analysis of both lterable and non-lterable
materials. Flow cytometric analysis gives near-real-time
See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 8.0 5.1.6. Alternative methods for control of microbiological quality
detection, but it is not as sensitive as solid phase cytometry.
To increase sensitivity for use in the pharmaceutical eld, it
often becomes necessary to add an incubation step in culture
media and in that case the method becomes a growth-based
method. Analysis of non-lterable samples may require serial
dilution to optimise performance, and particulate size can
have a signicant effect on performance. With the exception
of lterability, similar considerations apply to those of solid
phase cytometry. Clumping of bacteria can be a problem (e.g.
S. aureus).
Potential uses. In contrast with solid phase cytometry, this
method offers the potential to detect and enumerate the
microbial bioburden in materials containing signicant levels
of particulate matter. If a pre-incubation step is needed, the
method becomes a qualitative determination.
2-2-3. Direct epiuorescent ltration technique (DEFT)
Principles of measurement. This technique may be considered
to be a forerunner of solid phase cytometry. Micro-organisms
concentrated by ltration from the sample are stained with
a uorescent dye, formerly acridine orange and now more
commonly 4,6-diamidino-2-phenylindole (DAPI), that may
be detected by epiuorescent illumination. Fluorescent vital
staining techniques as employed in solid phase cytometry
(see 2-2-1.) are amenable to DEFT and uorescent redox dyes
such as 5-cyano-2,3-ditolyltetrazolium chloride (CTC) can be
used to highlight respiring cells. Coupled with microscopy,
the method allows rapid detection of micro-organisms,
the absolute sensitivity depending on the volume of
product ltered and the number of elds of view examined.
Semi-automated auto-focusing systems coupled to image
analysis have served to improve the utility of this method.
A modication to the principle employs sampling using an
adhesive sheet which permits collection of cells from surfaces,
staining on the sheet and subsequent direct observation under
the epiuorescence microscope.
Critical aspects. The distribution of micro-organisms on
the membrane affects method robustness. The intensity
of uorescence can be inuenced by the staining process
and the metabolic status of the micro-organisms. A brief
period of culture on the lter surface prior to staining
allows microcolony formation ; these microcolonies stain
readily, can be easily enumerated and are demonstrable
evidence of viability. Developments using uorescence in
situ hybridisation (FISH) arising from the complementary
interaction of a uorescently-labelled oligonucleotide probe
with a specic rRNA sequence offer a route to selective
detection.
Potential uses. DEFT is generally limited to low viscosity
uids although pre-dilution or pre-ltration has occasionally
been applied to viscous or particulate products. Bioburden
monitoring has been successfully achieved in aqueous
pharmaceuticals.
2-3. CELL COMPONENT ANALYSIS
2-3-1. Phenotypic
2-3-1-1. Immunological methods
Principles of measurement. Antibody-antigen reactions can be
employed to detect unique cellular determinants of specic
organisms. These reactions can be linked to agglutination
phenomena, colorimetric or uorimetric end-points offering
both quantitative and qualitative detection. Enzyme-linked
immunosorbent assays (ELISA) offer simple solid phase
methodologies.
Critical aspects. Immunological detection methods depend
upon the unique expression of specic identiers but
do not necessarily demonstrate the presence of viable
micro-organisms.
Potential uses. Detection and identication of specied
micro-organisms.
General Notices (1) apply to all monographs and other texts
2-3-1-2. Fatty acid profiles
Principles of measurement. The fatty acid composition of
micro-organisms is stable, well conserved and shows a high
degree of homogeneity within different taxonomic groups.
The isolate is grown on a standard medium and harvested.
The fatty acids are saponied, methylated and extracted and
the occurrence and amount of the resulting fatty acid methyl
esters are measured by high resolution gas chromatography.
The fatty acid composition of an unknown isolate is compared
with a database of known isolates for a possible match and
identication.
Critical aspects. The use of fatty acid proles for microbial
identication requires a high degree of standardisation. It
is critical for the fatty acid composition of microbial cells
that isolates are grown using standard media and standard
incubation conditions. Standard conditions for operation
of the gas chromatograph must be employed, with frequent
runs of calibration standards and known isolates being very
important.
Potential uses. Identication or characterisation of
environmental and product ora for contaminant tracing and
detection of specied micro-organisms.
2-3-1-3. Fourier transform infrared (FTIR) spectroscopy
Principles of measurement. A Fourier transformation of the
infrared spectrum of whole micro-organisms gives a stable,
recognisable pattern typical of the taxonomic groups of
micro-organisms. The analysis of the FTIR pattern can be
performed in instruments available on the market. The isolate
is grown on a standard medium and harvested. Cell mass is
transferred to a carrier, and the infrared spectrum is recorded.
The Fourier transformation is calculated and the pattern is
compared with a database of known isolates for a possible
match and identication.
Critical aspects. The use of FTIR-patterns for microbial
identication requires a high degree of standardisation. It is
critical for the FTIR-pattern of microbial cells that isolates
are grown using standard media and standard incubation
conditions. The cells must be in the same state of the growth
cycle when analysed. Particular attention needs to be paid to
the validation process.
Potential uses. Identication or characterisation of
environmental and product ora for contaminant tracing and
detection of specied micro-organisms.
2-3-1-4. Mass spectrometry
Principles of measurement. Gaseous breakdown products
released by heating microbial isolates in a vacuum can be
analysed by mass spectrometry, providing characteristic
spectra. Similarly, intact microbial cells, when subject to
intense ionisation under matrix-assisted laser desorption
ionisation-time of ight (MALDI-TOF) mass spectrometry,
release a distinctive pattern of charged species. Such spectra
can be compared with known proles as a rapid aid to
identication.
Critical aspects. Isolates require culture prior to analysis.
Potential uses. Identication or characterisation of
environmental and product ora for contaminant tracing and
detection of specied micro-organisms.
2-3-1-5. Biochemical assays based on physiological reactions
Principles of measurement. These assays are usually preceded
by a Gram stain or other early differentiation test to decide on
the appropriate testing protocol. Microbial cell suspensions are
tested using biochemical test kits. Micro-organisms are known
to have particular reactions to these biochemical substances,
e.g. utilisation of specic carbon sources. The identication
of the culture is done by comparing the biochemical reaction
prole with a database. These methods can be performed
manually or by automated instruments.
563
5.1.6. Alternative methods for control of microbiological quality
Critical aspects. A pure colony is needed which must not be
older than 3 days. The handling of the system is easy but the
interpretation of the results can be subjective. Depending on
the system used and the micro-organism under investigation,
the results can be available quickly.
Potential uses. Identication of environmental and product
ora for contaminant tracing and detection of specied
micro-organisms.
2-3-2. Genotypic
2-3-2-1. Nucleic acid amplification techniques (NAAT)
General principles of measurement. NAAT rely on the
reiteration of the process of DNA polymerisation, leading to
an exponential increase of a specic fragment of the nucleic
acid, i.e. the use of the polymerase chain reaction (PCR). In
this thermophilic cyclic process a specic DNA fragment is
amplied using oligonucleotide primers (see also general
method 2.6.21). RNA can also be amplied by PCR after
transcription into cDNA using a reverse transcriptase. This
technique is known as reverse transcriptase PCR (RT-PCR).
Alternatively, specic RNA-based amplication techniques,
for example nucleic acid sequence-based amplication
(NASBA) or transcription-mediated amplication (TMA)
are available to amplify multiple antisense copies of the RNA
target. Amplied nucleic acid fragments can be analysed by
several methods : fragment size analysis ; specic sequence
analysis ; reamplication with a second primer pair ; or
specic detection by hybridisation with a uorescent labelled
probe. Depending on the choice of analysis the amplication
technique can be qualitative, semi-quantitative or quantitative.
For identication/characterisation purposes sequence analysis
of specic parts of the genome can be used (i.e. 16S or 23S
rRNA targets).
General critical aspects. NAAT have many advantages over
classical methods for the detection of micro-organisms :
the methods are highly specic, provided that the primers
chosen are specic for a particular micro-organism or
group of micro-organisms ;
the procedures are rapid, overcoming the problem of
prolonged incubation times ;
the methods are highly sensitive allowing ideally the
detection and amplication of one single nucleic acid
fragment in the reaction mix.
However, there are numerous practical restrictions to its use :
the sensitivity of the methods is highly dependent on how
successfully the target fragments can be concentrated in
the sample ;
the presence of inhibitors of the enzymatic process result
in false negative reactions ;
the starting volume of the sample tested is small ;
the procedures are prone to cross-contamination from
previously amplied fragments resulting in false positive
results.
Depending on the aim, a choice must be made for amplication
of an RNA or DNA target. The target choice affects the
correlation with viability. The use of DNA as a marker has the
disadvantage that dead micro-organisms also contain DNA,
whereas mRNA is rapidly degraded in dead bacteria and is
considered a better marker for viability.
Critical aspects of RT-PCR. Reverse transcriptase-PCR is
characterised by the synthesis of cDNA using RNA as a
template. Reverse transcriptase is used for this step. A
specic part of the cDNA is subsequently amplied by
PCR. Depending on the quality of the RNA isolation, the
cDNA synthesis efciency can vary. RT-PCR can be used to
specically detect RNA if the DNA contamination of the RNA
sample is minimal.
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EUROPEAN PHARMACOPOEIA 8.0
Critical aspects of RNA amplification techniques. These
methods have proven to be very valuable for specic
(quantitative) RNA detection. However, they may be more
difcult to implement routinely.
Critical aspects of (semi-) quantitative detection (real-time
PCR). Classical PCR techniques are based on end-point
detection. In general fragment analysis is carried out using
agarose gels and specic size markers. However, there is no
correlation between the amount of PCR product at the end
of the reaction and the original amount of target molecule.
In contrast the amount of PCR product detected at the
beginning of the exponential phase of the reaction correlates
very well with the initial starting amount of nucleic acid.
Modern real-time PCR techniques are developed to measure
this exponential phase of the reaction. These techniques
generate amplication data from which the original amount
of target molecule can be deduced. A specic labelled probe
detects in real time the PCR product formed, allowing direct
visualisation of the exponential part of the PCR reaction.
By comparison with amplication plots of a standard
dilution series, a quantication of the target molecule can be
obtained. Automated real-time PCR systems are available
on the market. An additional advantage is that the chance
of cross-contamination is minimised, as PCR products are
scanned with a laser while the tubes remain closed. However,
generation of standards will be difcult to accomplish.
Critical aspects of amplification of genes coding for 16S or 23S
rRNA. A powerful application of PCR is the amplication
and subsequent sequence analysis of specic parts of the
genes coding for 16S or 23S rRNA. Analysis of these specic
DNA sequences allows in most cases the identication of
a micro-organism at species level. Selection of appropriate
universal primers, or even species-specic primer pairs, from
international databases allows a high specicity in fragment
amplication. Modern systematic classication is based on
comparative sequence analysis.
Potential uses. Owing to the high specicity of the
amplication techniques, they are very suitable for
identication purposes. NAAT are suitable for the detection
of specied micro-organisms or certain groups such as
mycoplasmas. Real-time quantitative PCR is needed for
enumeration.
2-3-2-2. Genetic fingerprinting
Principles of measurement. This technique characterises
and identies micro-organisms using restriction fragments
of nucleic acids from bacterial and fungal genomes. DNA
is extracted from a pure microbial cell lysate and cut into
fragments by restriction enzymes. DNA fragments are
size-separated by electrophoresis, visualised, and the pattern is
compared with other known patterns of microbial isolates. The
genetic ngerprint is a stable marker that provides denitive
species discrimination or even characterisation below species
level. Ribotyping is a typical example of this technique. There
are also ngerprinting methods based on PCR with primers
that bind to several sites in the microbial genome, creating
amplicons with a characteristic size distribution.
Critical aspects. There is a need for a pure colony, but
no preliminary cultivation step is necessary. The growth
conditions (temperature, type of media,) do not affect the
outcome of the analysis. For the identication of bacteria
semi-automated systems are on the market.
Potential uses. Genetic ngerprinting is more valuable for
strain discrimination (characterisation below species level)
than for identication of species.
3. GENERAL VALIDATION REQUIREMENTS
The purpose of this section is to provide guidance on the
validation of methods for use as alternatives to microbiological
methods of the Pharmacopoeia. For microbial recovery and
identication, microbiological testing laboratories sometimes
use alternative test methods to those described in the
See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 8.0 5.1.6. Alternative methods for control of microbiological quality
general chapters for a variety of reasons, such as economics,
throughput, and convenience. Validation of these methods
is required. Some guidance on validation is provided in the
General Notices section 1.1 on the use of alternative methods.
Validation of alternative microbiological methods must take
into account the large degree of variability associated with
conventional methods. When conducting microbiological
testing by conventional plate count, for example, one
frequently encounters a range of results that is broader than
ranges in commonly used chemical tests.
Where specic equipment is critical for the application of
the alternative method, the equipment, including computer
hardware and software, must be fully qualied as follows :
design qualication (DQ) to provide documented evidence
that the design of the equipment is suitable for correct
performance of the method ; to be provided by the supplier ;
installation qualication (IQ) to provide documented
evidence that the equipment has been provided and
installed in accordance with its specication ;
operational qualication (OQ) to provide documented
evidence that the installed equipment operates within
pre-determined limits when used in accordance with its
operational procedures ;
performance qualication (PQ) to provide documented
evidence that the equipment, as installed and operated
in accordance with operational procedures, consistently
performs in accordance with predetermined criteria
and thereby yields correct results for the method.
This is typically done with a model system (with test
micro-organisms) to make sure that the conditions used by
the user laboratory make it possible to satisfy the criteria
described by the supplier of the method in the laboratory.
Some alternative methods depend on the use of databases.
The extent of coverage of the database with respect to the
range of micro-organisms of interest must be taken into
account for validation purposes.
The value of a new or modied method must be demonstrated
in a comparative study between the ofcial method and the
alternative method. The characteristics dened in this chapter
must be used to establish this comparison.
3-1. TYPES OF MICROBIOLOGICAL TESTS
It is critical to the validation effort to identify the portion of
the test addressed by the alternative method. For example,
there are a variety of methods available to detect the presence
of viable cells. These methods may have applications in
a variety of tests (e.g. bioburden, sterility tests,) but may
not, in fact, replace the critical aspects of the test entirely.
For example, a sterility test by membrane ltration may be
performed according to the pharmacopoeial procedure up to
the point of combining the processed lter with the recovery
media, and after that the presence of viable cells might then
be demonstrated by use of some of the available methods.
Validation of this application would, therefore, require
validation of the recovery system employed rather than the
entire test.
General concerns. Validation of a microbiological method
is the process by which it is experimentally established
that the performance characteristics of the method meet
the requirements for the intended application. Since
microbiological tests have 3 basic applications, 3 separate
sets of validation criteria are required. These concerns are
described below.
3-2. VALIDATION OF ALTERNATIVE QUALITATIVE
TESTS FOR THE PRESENCE OR ABSENCE OF
MICRO-ORGANISMS
3-2-1. Accuracy and precision
A direct method to show the equivalence of 2 qualitative
methods would be to run them side by side and determine
the degree to which the method under evaluation shows
equivalence to the pharmacopoeial method. An example
General Notices (1) apply to all monographs and other texts
of this could be the sterility test where this would translate
into a comparison of the rate of positive and negative
results produced by the alternative method versus the
pharmacopoeial method for identical samples. However, in
a case such as the sterility test, the low number of failures
would required thousands of comparison tests to establish
equivalency and thus would be problematic.
A more feasible method for evaluating the precision
of an alternative qualitative method compared with a
pharmacopoeial method might be to observe the degree
of agreement between the two when the procedures are
performed repeatedly on different lots of the same product.
The accuracy and precision of the alternative method may be
expressed as the relative rates of false positive and false negative
results between the new method and the pharmacopoeial
method using a standardised, low-level inoculum.
The rate of occurrence of false negative results in the presence
of the sample for the 2 methods can be estimated using low
levels of test micro-organisms. This design is similar to the
standard bacteriostasis/fungistasis test ; however, the level of
micro-organisms inoculated must be very low, for example
about 5 CFU per unit. The level of inoculum should ensure a
frequency of failure rates high enough to provide a means to
compare the 2 methods. The alternative method must provide
at least as high a frequency of recovery as the pharmacopoeial
method.
3-2-2. Specicity
The specicity of an alternative qualitative method is its
ability to detect the required range of micro-organisms that
may be present in the sample under test. This concern is
adequately addressed by growth promotion of the media for
qualitative methods that rely upon growth to demonstrate
presence or absence of micro-organisms. For those methods
that do not require growth as an indicator of microbial
presence, the specicity assures that extraneous matter in the
test system does not interfere with the test. Where relevant
for the purpose of the test, mixtures of micro-organisms are
used during validation.
3-2-3. Limit of detection
The limit of detection of an alternative qualitative method
is the lowest number of micro-organisms in a sample that
can be detected under the stated experimental conditions. A
microbiological limit test determines the presence or absence
of micro-organisms. Due to the nature of microbiology, the
limit of detection refers to the number of micro-organisms
present in the original sample before any dilution or incubation
steps ; it does not refer to the number of micro-organisms
present at the time of testing.
The 2 methods (alternative and pharmacopoeial) must be
assessed by using an inoculum containing a low number of test
micro-organisms, for example about 5 CFU per unit, followed
by a measurement of recovery. The level of inoculation
must be adjusted until at least 50 per cent of the samples
show growth in the pharmacopoeial method. It is necessary
to repeat this determination several times, as the limit of
detection of a test is determined from an appropriate number
of replicates (for example not less than 5). The ability of the
2 methods to detect the presence of single organisms can be
demonstrated using the 2 test.
3-2-4. Robustness
The robustness of an alternative qualitative method is a
measure of its capacity to remain unaffected by small but
deliberate variations in method parameters, and provides an
indication of the methods reliability under a variety of normal
test conditions, such as different analysts, instruments, batches
of reagents and laboratories. Robustness can be dened as the
intrinsic resistance to the inuences exerted by operational and
environmental variables on the results of the microbiological
method. Robustness is a validation parameter best suited to
determination by the supplier of the method, but if critical
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5.1.6. Alternative methods for control of microbiological quality
parameters are modied by the user their effects on robustness
have to be evaluated. Robustness of a qualitative method is
judged by its ability to detect the test micro-organisms under
the deliberate variations to the method parameters.
3-3. VALIDATION OF ALTERNATIVE QUANTITATIVE
TESTS FOR ENUMERATION OF MICRO-ORGANISMS
3-3-1. Accuracy
The accuracy of an alternative quantitative method is the
closeness of the test results obtained by the alternative
method to the value obtained by the pharmacopoeial method.
Accuracy must be demonstrated across the practical range of
the test. Accuracy is usually expressed as the percentage of
recovery of micro-organisms by the method.
Accuracy may be shown by preparing a suspension of
micro-organisms at the upper end of the range of the test,
serially diluted down to the lower end of the range of the test.
For example, if the alternative method is meant to replace
the traditional plate count method for viable counts, then a
reasonable range might be 100-106 CFU per mL. If it is, instead,
a replacement for the MPN method, a much more narrow
range may be used. At least 5 suspensions across the range
of the test must be analysed for each test micro-organism. If
the alternative method is meant to replace the conventional
method, it must provide an estimate of viable micro-organisms
of not less than 70 per cent of the estimate provided by the
pharmacopoeial method.
The protocol used to check the linearity (see 3-3-5.) of
the method may also be used to check the accuracy :
the suspensions of micro-organisms prepared for the
alternative method are counted at the same time using the
pharmacopoeial method. Accuracy is demonstrated if the
suitability tests show that the slope of the regression line does
not differ signicantly from 1 and if the y-intercept is not
signicantly different from 0.
3-3-2. Precision
The precision of an alternative quantitative method is the
degree of agreement among individual test results when the
procedure is applied repeatedly to multiple samplings of
homogeneous suspensions of micro-organisms under the
prescribed conditions. The precision is usually expressed as
the variance, standard deviation or coefcient of variation of a
series of measurements.
At the very least, a suspension of micro-organisms with a
concentration usually in the middle of the range is counted
several times. The number of replicates is chosen so that
the entire test can be carried out during the same working
session, i.e. under the same operating conditions and without
any change in the suspension of micro-organisms. Other
working sessions are then carried out under conditions of
maximum variability (different reagents, different operators,
different days, etc.). The variance of the results observed in
each of the working sessions (groups) is calculated. If the
variances are homogeneous, the variance of the repeatability
can be calculated. The inter-group variance of the results is
calculated. The variance of the intermediate precision is the
sum of the variance of the repeatability and the inter-group
variance. The coefcients of variation are then calculated.
Generally, a coefcient of variation in the 10-15 per cent range
is acceptable. Irrespective of the specic results, the alternative
method must have a coefcient of variation that is not larger
than that of the pharmacopoeial method.
3-3-3. Specicity
The specicity of an alternative quantitative method is
demonstrated using a range of appropriate micro-organisms.
Where relevant for the purpose of the test, mixtures of
micro-organisms are used during validation.
3-3-4. Limit of quantication
The limit of quantication of an alternative quantitative
method is the lowest number of micro-organisms that can be
accurately counted. As it is not possible to obtain a reliable
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EUROPEAN PHARMACOPOEIA 8.0
sample containing a known number of micro-organisms, it is
essential that the quantication limit is determined from a
number of replicates, for example at least 5. The results of
the linearity and accuracy studies can also be used. Here,
the lowest concentration in the linear range is considered to
be the limit of quantication of the method. The limit of
quantication must not be a number greater than that of the
pharmacopoeial method.
3-3-5. Linearity
The linearity of an alternative quantitative method is its ability
to produce results that are proportional to the concentration
of micro-organisms present in the sample within a given
range. The linearity must be determined over the range
corresponding to the purpose of the alternative method.
A method to determine this would be to select different
concentrations of each test micro-organism and conduct
several replicates of each concentration. The number of
replicates is chosen so that the entire test can be carried out
during the same working session. 2 more working sessions
are then completed under conditions of maximum variability
(different reagents, different operators, different days, etc.).
After checking the homogeneity of the variances of the
results obtained for each concentration, the regression line is
calculated. Linearity is demonstrated if the estimated slope
is signicant and if the test for deviation from linearity is
non-signicant.
3-3-6. Range
The range of an alternative quantitative method is the interval
between the upper and lower levels of micro-organisms that
have been determined with precision, accuracy, and linearity
using the method as written. The range is determined from
studies of precision, accuracy and linearity.
3-3-7. Robustness
The robustness of an alternative quantitative method is a
measure of its capacity to remain unaffected by small but
deliberate variations in method parameters and provides an
indication of its reliability under a variety of normal test
conditions, such as different analysts, instruments, batches of
reagents and laboratories. Robustness can be dened as the
intrinsic resistance to the inuences exerted by operational and
environmental variables on the results of the microbiological
method. Robustness is a validation parameter best suited to
determination by the supplier of the method, but if critical
parameters are modied by the user their effects on robustness
have to be evaluated. Robustness of a quantitative method is
judged by its ability to enumerate with statistical relevance
the test micro-organisms under the deliberate variations to
the method parameters.
3-4. VALIDATION OF ALTERNATIVE IDENTIFICATION
TESTS
There is a large body of evidence that different methods vary
considerably in their ability to identify micro-organisms in
pharmacopoeial products. It must be accepted that a method
of systematics needs to be internally consistent, but may
differ from others in identication of isolates. In other words,
identication of an isolate based on biochemical activity may
lead to one conclusion, identication by fatty acid analysis
to another, identication by DNA analysis may lead to a
third, and other methods may lead to alternative conclusions.
Microbiological identications by a particular system ow
directly from previous experience with that system, and
therefore may well differ from identications by another
system. It is critical that each system provides a consistent
identication of isolates from pharmacopoeial products.
3-4-1. Accuracy
The accuracy of an alternative identication method is
its ability to identify the desired micro-organism to the
required taxonomic level and to differentiate it from
other micro-organisms present in the sample. It must be
demonstrated with a series of test micro-organisms or
See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 8.0 5.1.6. Alternative methods for control of microbiological quality
micro-organisms obtained from a typical sample previously
identied by another method.
3-4-2. Precision
The precision of an alternative identication method is the
degree of agreement among individual test results when the
procedure is applied repeatedly to multiple samplings of
suspensions of test micro-organisms across the range of the
test.
3-4-3. Robustness
The robustness of an alternative identication method is a
measure of its capacity to remain unaffected by small but
deliberate variations in method parameters, and provides
an indication of its reliability under a variety of normal
test conditions such as different analysts, instruments,
batches of reagents and laboratories. Robustness can be
dened as the intrinsic resistance to the inuences exerted
by operational and environmental variables on the results
of the microbiological method. Robustness is a validation
parameter best suited to determination by the supplier of the
method, but if critical parameters are modied by the user
their effects on robustness have to be evaluated. Robustness
of an identication method is judged by its ability to identify
consistently the test micro-organisms under the deliberate
variations to the method parameters.
4. SPECIFIC VALIDATION REQUIREMENTS
4-1. BACKGROUND
Validation is dened in various contexts with some differences,
but a consensus denition is to establish documented evidence
that a process will consistently achieve what it is intended
to do. Hence, in order to perform correct validation of a
new method it is critical to understand and dene what the
procedure is intended to achieve.
2 levels of validation must be envisaged for the application
of conventional or alternative microbiological methods.
Primary validation of a method is typically performed by
the supplier of the new method, whereas validation for the
actual intended use, which is a verication of the suitability
or applicability of the method in a given situation, must be
seen as the responsibility of the user. Before validation for the
actual intended use, performance qualication is carried out
by the user as described in 3. General validation requirements.
Typically, microbiological methods use specic characteristics
of micro-organisms as indicators or detection principles for
more general questions. The information needed is presence,
number, viability, resistance or identity of micro-organisms in
a given product or environment.
A given method will usually give an indirect and conditional
answer to the questions. For example, the total number and
viability of micro-organisms is indicated by the number of
micro-organisms able to reproduce under a certain set of
conditions for sample preparation, cultivation and incubation.
Reproduction in classical microbiology is hence taken as the
general indicator for viability. There are other parameters,
however, that can be used as an indication of viability. The
level of ATP or accumulation or metabolism of substrates
in living cells can also be taken as an indicator for viability.
The results of different indication methods for viability may
not always be identical. Micro-organisms may not be able to
reproduce on a given medium, but may still accumulate and
metabolise a substrate. Micro-organisms may be unable at a
given state of damage to accumulate a substrate, but may still
be able to recover and to reproduce.
Another example is the various methods used for identication
of micro-organisms. Characterisation of the metabolic
pattern of micro-organisms is frequently used for species
identication, whereas another method consists of the
comparison of DNA sequences. Again, the answer obtained
may not be fully coincident for the different identication
methods, and while one answer may be appropriate for the
General Notices (1) apply to all monographs and other texts
construction of a correct phylogenetic correlation tree, another
answer may be more useful in the context of pathogenicity or
other properties of the differentiated micro-organisms.
4-1-1. Primary validation
In order to characterise a specic microbiological method,
the principle of detection must be clearly described by the
supplier. The method must be fully detailed with respect to
the conditions required for application, the materials and
equipment needed, and the expected signal. The application
principle should be described in a peer-reviewed journal.
The principle of detection must be characterised in a model
system and/or with a panel of test micro-organisms, by at least :
prerequisite treatment of sample or micro-organisms ;
type of response ;
specicity of the response ;
limit of detection ;
range ;
linearity of the response ;
accuracy and precision of the response ;
robustness of the method in a model system ;
limits of suitability.
Once the method has been characterised in this way by the
supplier, the principle of detection need not be veried by
each user.
4-1-2. Validation of alternative microbiological method
4-1-2-1. Risk-benefit analysis
For validation of specic alternative microbiological methods
it is critical that the purpose of the procedure is precisely
outlined. Based on the purpose, the type and depth of
information needed must be dened. The information
obtained by, and the limitations of, the conventional method
and the alternative method must be considered and compared
in a risk-benet analysis.
An alternative method can be justied as being applicable if
the information obtained gives a scientically sound answer
to the questions asked in the procedure, and if the limitations
of the method are not more severe than the limitations of the
conventional method.
4-1-2-2. Validation for the actual intended use
The alternative method must be applied in the procedure used
and with the samples to be analysed under the responsibility
of the user, and must be shown to give comparable results
as characterised in a model system by the supplier. Specic
questions to be asked where applicable are :
compatibility of the response with the sample preparation
needed for product testing ;
limit and range of detection of the method with regard to
sample size and sample availability ;
specicity of the response with regard to the inuence of
the ingredients of the product ;
linearity of the response with regard to all types of samples
to be analysed ;
accuracy and precision of the response with regard to all
types of samples to be analysed ;
robustness of the method with regard to all types of samples
to be analysed.
Acceptance criteria for the method in routine use will need
to be dened as a function of the application and of the
validation data.
4-2. BIOLUMINESCENCE FOR ENUMERATION OF
MICRO-ORGANISMS
4-2-1. Risk-benet analysis
Extensive scientic evidence and use for years supports the
capability of the ATP viability marker to detect the same
range of micro-organisms as is encountered using standard
plating methods. Since this method is growth-dependent,
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5.1.6. Alternative methods for control of microbiological quality
the improvement comparing to the plating methods is the
rapidity to obtain a result (from 5 days with the plating
methods to 24 h for bioluminescence). It is possible to identify
the bioluminescence-detected micro-organisms from the
incubation step medium, but it has to be remembered that
in a mixed culture some micro-organisms may out-compete
others during incubation. This method provides evaluation of
samples within 24 h for lterable and non-lterable products
(water, in-process control, environmental samples, solid and
liquid raw materials, solid and liquid nished products, etc.)
and for a large number of samples, when the detection step
is automated.
4-2-2. Validation for the actual intended use
The method relies upon the detection of ATP from viable
micro-organisms. Performance qualication is carried out
with test micro-organisms to make sure that under the
conditions applied by the user laboratory it is possible to
satisfy the criteria described by the supplier for precision,
accuracy and linearity (quantitative method), or limit of
detection (qualitative and semi-quantitative method) over
the range required for the intended use. Following this step,
validation proceeds in 3 phases :
phase 1 : fertility of the medium in the presence of the
product (if an incubation step is performed) ;
phase 2 : search for interferences that may increase or inhibit
the ATP production (by addition of an ATP standard
solution to the product to test) ;
phase 3 : comparative testing with the pharmacopoeial
method.
A detailed example of validation of the bioluminescence
method is given at the end of this chapter.
4-3. CYTOMETRY (SOLID AND FLOW) FOR
ENUMERATION OF MICRO-ORGANISMS
4-3-1. Risk-benet analysis
Extensive scientic evidence supports the capability of
this uorescence viability marker to detect and/or count
a wider range of micro-organisms than are encountered
using standard plating methods. Cytometry will detect all
viable micro-organisms including some that may not be
discernable by growth-based methods. Whilst being rapid, the
recovery of micro-organisms post-analysis is limited. Thus
the further processing of analysed samples for identication
would require alternative uorescent stains or an alternative
method. Currently it is not possible to use this method for
routine identication of micro-organisms, although basic
morphology is readily discernable in solid phase cytometry
under uorescent microscopes. This method provides rapid
evaluation of samples and hence allows for a proactive
approach to pharmaceutical manufacturing, facilitating
building quality into pharmaceutical operations. This method
is not growth-dependent and hence all metabolically active
micro-organisms will be detected. However, the limit of
detection for ow cytometry is currently such that it cannot
be used for enumeration by direct examination for most
pharmaceutical samples. If pre-incubation is necessary, the
estimation becomes semi-quantitative (limit test).
4-3-2. Validation for the actual intended use
The method relies upon the detection of a uorescent signal
from labelled micro-organisms.
Performance qualication is carried out to ensure that
the instruments perform within their dened operational
parameters. This involves the use of uorescent standards of
prescribed intensity and cultures of known type and number
of micro-organisms. These tests challenge the quantitative
detection system. Reagents and consumables (negative
controls) must also be utilised to ensure that the routine test
protocol is applicable, and that the quality of the materials
used in the test do not contribute to the nal result. Pure
culture experiments involving test micro-organisms are used
to challenge the detection system, and to compare test results
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EUROPEAN PHARMACOPOEIA 8.0
with those obtained using standard plate count. Multiple
replicates (at least 5) from overnight cultures diluted across a
concentration range (e.g. 100 per cent, 75 per cent, 50 per cent,
25 per cent and 10 per cent) must be used to evaluate linearity,
accuracy, precision, range, specicity, limit of quantication
(quantitative method) and limit of detection (ow cytometry
with pre-incubation step). Since cytometry has high sensitivity
(solid phase cytometry can detect single cells, whereas ow
cytometry is sensitive to a level of around 10-50 cells per
millilitre), and detection is not growth based, the linearity of
the instrumentation can be tested by comparison of the actual
results with the expected value.
Following this step, validation proceeds in 2 phases : validation
with respect to the product to be examined and comparative
testing. Results of each phase must be evaluated against
pre-determined acceptance criteria using positive and negative
controls :
phase 1 : individual materials to be evaluated by cytometry
must be spiked with a dened level of micro-organisms to
ensure that the sample preparation process and the samples
themselves do not have an impact upon the performance
of the detection system ; specically, the sample matrix
must not affect detection (i.e. contain endogenous
chromophores, auto-uorescent particles), and in the case
of ow cytometry, sample size/dilution and ow rate must
be determined for optimal performance ;
phase 2 : testing must be performed in which the results
obtained by cytometry and the pharmacopoeial method are
compared ; the number of samples and the testing period
must be dened in a comparability protocol ; the number
of samples required will vary, but must be representative of
the material evaluation process (i.e. time/number), and
must allow for statistical evaluation ; all samples must be
prepared according to dened procedures and evaluated
against selected validation and acceptance criteria, similar
to those used for pure culture evaluation.
4-4. FATTY ACID PROFILES FOR IDENTIFICATION
4-4-1. Risk-benet analysis
Identication by fatty acid proles may be more precise
than the identication methods based on metabolic proles
in conventional microbiological culture methods. The
database is broader than for conventional culture methods.
Pre-incubation is needed, but extraction and identication is
faster than in biochemical methods and hence, the result is
obtained faster. Other modern methods, such as 16S rRNA
sequence analysis or genetic ngerprinting, have a similar
broad differentiation range and give a result as fast as this
method.
Separation of closely related micro-organisms (e.g. E. coli
and Salmonella spp.) can be difcult by fatty acid proles.
Where the identication of closely related micro-organisms
is especially important, other systems may give more precise
results. For a given application it is important to specify which
types of micro-organisms are most important to be identied.
If it is most critical to characterise the correct phylogenetic
species of the isolate, DNA sequence-based identication
methods will give more reliable results.
Limitations of identication by fatty acid proles are also seen
in the necessity to grow micro-organisms on standardised
media under standard temperature conditions and durations
of incubation. Micro-organisms that cannot be cultivated on
such media cannot be identied.
4-4-2. Validation for the actual intended use
Using a range of test micro-organisms and at least 3 replicate
determinations in each case, it must be demonstrated that the
method yields consistent results.
A signicant number of isolates from typical samples to be
analysed by the user must be identied, at least 3 times each.
The results in each case should be consistent and in accord
with those obtained using alternative identication methods.
See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 8.0 5.1.6. Alternative methods for control of microbiological quality
Where a different identication result is found in another
identication system, the reason for the difference must be
investigated. Where a scientically plausible explanation exists
for the recognition of a different species, a difference between
identication systems may be acceptable. In such a case it
must be assured that the recognition of the identied species
is robust. It must also be assured that the system does not
group poorly recognised isolates under one species thereby
simulating the repeated isolation of a single species.
4-5. NUCLEIC ACID AMPLIFICATION TECHNIQUES
4-5-1. Risk-benet analysis
NAAT are widely used in diagnostics for their precision and
rapidity at a relatively low cost (for the analysis, but not for the
instruments,) when compared with the traditional methods.
Provided that specic validations have been performed, when
NAAT are appropriately used, they may offer advantages
in some elds in comparison to classical methods ; on the
other hand classical methods are generally more easily
standardisable, need a lower level of technical competence and
may have lower costs. Even when NAAT are not more difcult
to perform than traditional methods, the interpretation of the
results generally needs a high degree of scientic competence.
When used for identication, DNA-based methods cannot
discriminate between dead and live micro-organisms. That
means that they cannot be directly used on the product
but only after passage on a traditional culture medium,
thereby losing part of the advantage in rapidity. Moreover,
if used directly on the product at the end of the analysis,
these methods do not result in a strain to be used in
further experiments and may not give advantages when the
micro-organisms to be detected are poorly cultivable or
stressed. RNA amplication techniques (e.g. RT-PCR) may
identify living micro-organisms (but not spores) directly in
the products, but in comparison to traditional methods are
much more difcult to use routinely. On the other hand,
where specic primers are used, identication (or typing) by
NAAT is more precise than the traditional methods and in
some cases may have other advantages : for instance for the
identication of some vaccines (e.g. cholera vaccine, whole
cell pertussis vaccine,) their use may substitute for that of
specic sera and contribute to reducing the use of animals, or
may give a very specic identication where this is presently
lacking (e.g. BCG vaccine).
These methods are in general non-quantitative (PCR) or
semi-quantitative (real-time PCR), meaning that their results
cannot be compared with those of a colony count where an
exact enumeration of the micro-organisms present in the
sample is requested, but even if colony count has a valence
consolidated in time this dogma may not be veried for
bacteria which have a tendency to clump (mycobacteria) or are
organised in chains or in clusters (streptococci, staphylococci),
therefore an accurate standardisation of the semi-quantitative
methods may give results of comparable reliability.
4-5-2. Validation for the actual intended use
The method is validated according to chapter 2.6.21.
Comparison of conventional and PCR-based methodologies,
which differ in sensitivity and specicity, is particularly
difcult and may lead to divergent conclusions.
The following example is published for information and not
for general application.
Example validation of an alternative method :
detailed protocol followed by a laboratory
for the implementation of bioluminescence
BACKGROUND
Methods using a pre-incubation step in liquid medium
(bioluminescence in tube or microtitre plate) do not
offer quantitative information but a presence/absence
General Notices (1) apply to all monographs and other texts
determination in the quantity analysed. Using more than a
single sample quantity, the system may offer semi-quantitative
determination (limit test). For example, the classical tested
quantity for viable aerobic count on non-sterile products is
0.1 g or 0.1 mL leading to absence in 0.1 g or 0.1 mL, i.e.
less than 10 micro-organisms in 1 g or 1 mL for a negative
result and more than or equal to 10 micro-organisms in 1 g
or 1 mL in case of a positive result. If 0.01 g or 0.01 mL is
tested simultaneously, a negative result corresponds to a
number of micro-organisms less than 100 in 1 g or 1 mL.
The combination between negative for 0.01 g or 0.01 mL
and positive for 0.1 g or 0.1 mL permits an estimate of the
contamination level of the product to be less than 100 but more
than or equal to 10 micro-organisms in 1 g or 1 mL.
As mentioned in section 2., bioluminescence can be used as
a quantitative method if micro-organisms are captured on a
ltration membrane and later incubated in culture medium
(bioluminescence on membrane).
The protocol below describes validation aspects for qualitative,
semi-quantitative and quantitative methods.
PERFORMANCE QUALIFICATION OF THE
ALTERNATIVE METHOD
Specicity
Screen the method with test micro-organisms appropriate to
the method. For example, for microbial aerobic viable count
on non-sterile products, use at least the micro-organisms
described in chapter 2.6.12 for the fertility of the media in the
presence of product. This determination is performed at least
3 times with each micro-organism. Acceptance criterion : all
test micro-organisms are successfully detected.
Limit of detection (only for semi-quantitative or qualitative
methods)
Prepare a low inoculum (about 5 CFU in the initial sample)
of each test micro-organism. Perform the analysis in at
least 5 replicates with the pharmacopoeial method and
with the bioluminescence method concerned. Acceptance
criterion : the ability of the 2 methods to detect the presence
of a single micro-organism can be demonstrated using the
2 test. Alternative procedure : prepare a series of dilutions
of micro-organisms to have a count in the next dilution
of about 5 CFU per inoculum (e.g. : 10 CFU/inoculum,
5 CFU/inoculum, 2.5 CFU/inoculum, 1.25 CFU/inoculum,
0.75 CFU/inoculum). Perform the test on 5 independent series
of dilutions with the pharmacopoeial method and with the
bioluminescence method concerned. Determine the limit of
detection for each method. It corresponds to the last dilution
where the result is positive for the 5 series. Acceptance
criterion : the limit of detection of the bioluminescence
method is equal to or lower than that of the pharmacopoeial
method.
Limit of quantication (quantitative method)
This can be performed at the same time as the linearity
determination. It corresponds to the lowest concentration
of the chosen range that satises the criteria for linearity,
accuracy and precision. Acceptance criterion : the limit of
quantication of the bioluminescence method is equal to or
lower than that of the pharmacopoeial method.
Precision
Quantitative evaluation. For each test micro-organism,
perform at least 5 replicates during the same series including
at least the concentration of micro-organisms corresponding
to the middle of the range. Perform 3 independent tests.
Carry out a statistical analysis to compare the precision of
the 2 methods or calculate the coefcient of variation (CV).
Acceptance criterion : CV 15 per cent to 30 per cent or
precision not different with the risk alpha equal to 5 per
cent between the 2 methods. If precision is different, the
bioluminescence method is better than the pharmacopoeial
method, indicated by a smaller standard deviation.
569
5.1.6. Alternative methods for control of microbiological quality
Qualitative or semi-quantitative evaluation. Use the alternative
procedure described for setting the limit of detection and
report the frequency of positive results in parallel with the
pharmacopoeial method. Acceptance criterion : the frequency
of positive results at the detection limit is 100 per cent and
this frequency is better than or equal to the pharmacopoeial
method.
Linearity
For each test micro-organism, prepare 5 concentrations in
the range of the bioluminescence method (range is normally
indicated by the supplier). Perform the pharmacopoeial and
the bioluminescence methods in parallel. Repeat this test
2 further times to have results on 3 independent tests. Test
for linear regression, presence of a slope, and lack of t with
the F test at alpha equal to 5 per cent. If statistical analysis is
not possible, calculate the correlation coefcient (r2) and the
slope between the 2 methods. Acceptance criterion : statistical
analysis may show linear regression, the presence of a slope
and no lack of t with a risk of 5 per cent. Equation y = a + bx
is determined where b is the slope and a the intercept. If
no statistical analysis is available, r2 is at least 0.9 and the
slope does not diverge by more than 20 per cent from 1 (b
between 0.8 and 1.2). If the linearity is not demonstrated in
such a large range, the range can be decreased and linearity
demonstrated with only 3 concentrations in place of 5.
Accuracy
Quantitative evaluation. Accuracy can be determined with
data obtained in linearity. For each micro-organism use
3 to 5 concentrations within the linear range of the method.
Perform statistical analysis (Students t test at risk 5 per cent)
to test the conformity of the estimated slope (value = 1)
versus the obtained slope and to test the conformity of
the estimated intercept (value = 0) versus the obtained
intercept. For example, if the estimated slope is b with a
standard deviation s(b) of 0.090 with 5 concentrations of
micro-organisms, calculate t = (b 1)/s(b). For intercept a,
with standard deviation equal to s(a), t = (a 0)/s(a). Compare
these values to the Students t at 5 per cent, for 13 degrees of
freedom (3 tests, 5 concentrations). Acceptance criterion : if
the t values obtained are less than the Students t, the method
is exact in the applied range. In the case that there is no
conformity for the slope (slope different from 1) or for the
intercept (intercept different from 0) the method is not exact
over the applied range.
Qualitative or semi-quantitative evaluations. Use the
alternative procedure described for setting the limit of
detection. Calculate the proportion of false negatives for
bioluminescence and for the pharmacopoeial method over
all tested dilutions. Compare the extent of false negatives
for the 2 or 3 concentrations of micro-organisms just
under the detection limit (for example 5 CFU/inoculum,
2.5 CFU/inoculum or 1.25 CFU/inoculum) giving a positive
result. By denition, the detection limit corresponds to 0 per
cent of false negatives. Acceptance criterion : the percentage
of false negatives for the bioluminescence method at sample
concentrations below the detection limit must be equal to or
lower than that of the pharmacopoeial method.
Range
This is the interval between the lowest and the highest
concentrations of micro-organisms where linearity, precision
and accuracy have been demonstrated.
Robustness
The information is given by the supplier.
VALIDATION FOR THE ACTUAL INTENDED USE
In the example given, there was no need to determine the
accuracy and detection limit in the presence of the product.
The validation consists of 3 parts, verifying :
phase 1 : the fertility of the medium in the presence of the
product ;
570
EUROPEAN PHARMACOPOEIA 8.0
phase 2 : the absence of interference from the product that
may increase or inhibit ATP production ;
phase 3 : the testing of the product in parallel with the
pharmacopoeial method.
These 3 parts of validation are performed on 3 independent
tests using for example at least 2 different batches of product.
Phase 1 : fertility of the medium in the presence of the
product
If the product has a known high contamination level (more
than 500 micro-organisms per gram or millilitre) the
incubation step is unnecessary, the micro-organisms can
be detected directly. In this case testing the fertility of the
medium in the presence of the product is not necessary.
However, pharmaceutical products are generally contaminated
at a much lower level and growth of the micro-organism is
necessary to obtain detection with bioluminescence. It must
therefore be proven that the product does not inhibit the
growth of micro-organisms under the conditions of the test.
In order to do so, separately add inoculum at not more than
100 CFU for each test micro-organism into the portion of
medium containing the product. For bioluminescence in
tube or microtitre plate, perform the bioluminescence test.
For bioluminescence on membrane, incubate at 30-35 C or
20-25 C for 5 days and count the bioluminescent colonies
on the membrane. Acceptance criterion : the test is positive
(bioluminescence in tube or microtitre plate); the quantitative
recovery of the micro-organism is at least 70 per cent
(bioluminescence on membrane).
Phase 2 : search for interference of the product
The objective is to show that the product does not add stray
light or non-microbial ATP (does not lead to false positive
result : criterion A) or does not decrease the ATP detection
(does not lead to a false negative result : criterion B).
Bioluminescence in tube or microtitre plate
A. Perform the bioluminescence test with the culture broth
alone and with the culture broth in the presence of the
product. Determine the RLU value for culture broth alone
and the RLU value for culture broth in the presence of
product.
B. Perform the bioluminescence test with the culture broth
alone and the culture broth in the presence of ATP.
Determine the response coefcient for ATP concentration
in per cent.
Acceptance criterion :
criterion A : the RLU value of culture broth in the presence
of product is less than twice the RLU value of culture
broth alone (if criterion A is not satised, it is necessary to
determine a specic threshold for this product);
criterion B : the RLU value of culture broth in the presence
of product and ATP is within the interval 25 per cent
to 200 per cent of the RLU value of culture broth in the
presence of ATP.
Bioluminescence on membrane : perform the complete
bioluminescence test to search for interference. Acceptance
criterion : the recovery of micro-organisms is greater than or
equal to 70 per cent and not more than 200 per cent.
Phase 3 : analysis of the product in parallel with the
pharmacopoeial method
Perform the test according to the validated method for the
product concerned in parallel with the pharmacopoeial
method to show the relationship between the 2 methods for
the product concerned, on 3 independent tests and using
at least 2 different batches. Express the result as positive or
negative in a certain quantity (bioluminescence in tube or
microtitre plate) or express the count per ltered quantity
(bioluminescence on membrane). Acceptance criterion :
results must be correlated with the pharmacopoeial method.
See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 8.0 5.1.8. Microbiological quality of herbal medicinal products and extracts

01/2008:50107 The risk assessment can be based mainly on the manufacturing

conditions if these include rigorous inactivation steps (for
example, for gelatin etc., and products terminally sterilised by
5.1.7. VIRAL SAFETY steam or dry heat as described in the general texts on sterility
(5.1)).
This chapter provides general requirements concerning the
viral safety of medicinal products whose manufacture has
involved the use of materials of human or animal origin. 01/2014:50108
Since viral safety is a complex issue, it is important that a
risk assessment is carried out. Requirements to be applied to 5.1.8. MICROBIOLOGICAL QUALITY
a specic medicinal product are decided by the competent
authority. OF HERBAL MEDICINAL PRODUCTS
Where the risk of viral contamination exists, complementary FOR ORAL USE AND EXTRACTS USED
measures are used as appropriate to assure the viral safety of IN THEIR PREPARATION
medicinal products, based on :
selection of source materials and testing for viral This general chapter presents recommended acceptance criteria
contaminants ; for the microbiological quality of both herbal medicinal
products for oral use and the extracts that are used in their
testing the capacity of the production process to remove preparation.
and/or inactivate viruses ;
testing for viral contamination at appropriate stages of Microbial examination of non-sterile products is performed
production. according to the methods given in general chapters 2.6.12,
Where appropriate, one or more validated procedures for 2.6.13 and 2.6.31. Acceptance criteria based upon the total
removal or inactivation of viruses are applied. aerobic microbial count (TAMC) and the total combined
yeasts/moulds count (TYMC) are given below.
Further detailed recommendations on viral safety, including
Acceptance criteria are based on individual results or on
validation studies, are provided, in particular, by the Note for
the average of replicate counts when replicate counts are
guidance on virus validation studies : the design, contribution
performed (e.g. direct plating methods).
and interpretation of studies validating the inactivation and
removal of viruses (CPMP/BWP/268/95) of the Committee for A list of specied micro-organisms for which acceptance
Proprietary Medicinal Products, and the ICH guideline Q5A : criteria are set can be found below. The list is not necessarily
Viral safety evaluation of biotechnology products derived from exhaustive and for a given preparation it may be necessary
cell lines of human or animal origin (including any subsequent to test for other micro-organisms depending on the nature
revisions of these documents). of the starting materials, the manufacturing process and the
intended use.
Requirements concerning immunological products for
veterinary use are dealt with in the monographs Vaccines HERBAL MEDICINAL PRODUCTS
for veterinary use (0062) and Immunosera for veterinary use A. Herbal medicinal products containing herbal drugs,
(0030) and related general chapters. with or without excipients, intended for the preparation of
Risk assessment infusions and decoctions using boiling water (for example
herbal teas, with or without added avourings)
A risk assessment with respect to viral safety is carried out
where materials of human or animal origin are used as TAMC (2.6.12) Acceptance criterion : 107 CFU/g
ingredients of medicinal products or in the manufacture of Maximum acceptable count : 50 000 000 CFU/g
active substances, excipients or medicinal products. TYMC (2.6.12) Acceptance criterion : 105 CFU/g
The principle of the risk assessment is to consider various Maximum acceptable count : 500 000 CFU/g
factors that may inuence the potential level of infectious Escherichia coli Acceptance criterion : 103 CFU/g
particles in the medicinal product and factors related to the (2.6.31)
use of the medicinal product that determine or inuence the Salmonella (2.6.31) Absence (25 g)
viral risk to the recipients.
B. Herbal medicinal products containing, for example,
The risk assessment takes into consideration relevant factors, extracts and/or herbal drugs, with or without excipients,
for example : where the method of processing (for example, extraction)
the species of origin ; or, where appropriate, in the case of herbal drugs, of
pre-treatment reduces the levels of organisms to below
the organ, tissue, uid of origin ; those stated for this category
the potential contaminants in view of the origin of the
TAMC (2.6.12) Acceptance criterion : 104 CFU/g or CFU/mL
raw material and the history of the donor(s), preferably
Maximum acceptable count : 50 000 CFU/g
including epidemiological data ; or CFU/mL
the potential contaminants from the manufacturing TYMC (2.6.12) Acceptance criterion : 102 CFU/g or CFU/mL
process (for example, from risk materials used during Maximum acceptable count : 500 CFU/g
manufacture); or CFU/mL
the infectivity and pathogenicity of the potential Bile-tolerant
gram-negative Acceptance criterion : 102 CFU/g or CFU/mL
contaminants for the intended recipients of the medicinal bacteria (2.6.31)
product, taking account of the route of administration of Escherichia coli
the medicinal product ; Absence (1 g or 1 mL)
(2.6.31)
the amount of material used to produce a dose of medicinal Salmonella (2.6.31) Absence (25 g or 25 mL)
product ;
C. Herbal medicinal products containing, for example,
controls carried out on the donor(s), on the raw material, extracts and/or herbal drugs, with or without excipients,
during production and on the nal product ; where it can be demonstrated that the method of processing
the manufacturing process of the product and its capacity (for example, extraction with low-strength ethanol or water
to remove and/or inactivate viruses. that is not boiling, or low-temperature concentration)

General Notices (1) apply to all monographs and other texts 571
5.1.9. Guidelines for using the test for sterility EUROPEAN PHARMACOPOEIA 8.0

or, in the case of herbal drugs, of pre-treatment, would and the efciency of the adopted sampling plan. Hence for
not reduce the level of organisms sufciently to reach the the purpose of this text a batch is dened as a homogeneous
criteria required under B collection of sealed containers prepared in such a manner that
the risk of contamination is the same for each of the units
TAMC (2.6.12) Acceptance criterion : 105 CFU/g or CFU/mL
contained therein.
Maximum acceptable count : 500 000 CFU/g
or CFU/mL In the case of terminally sterilised products, physical proofs,
TYMC (2.6.12) Acceptance criterion : 104 CFU/g or CFU/mL biologically based and automatically documented, showing
Maximum acceptable count : 50 000 CFU/g correct treatment throughout the batch during sterilisation are
or CFU/mL of greater assurance than the sterility test. The circumstances
Bile-tolerant in which parametric release may be considered appropriate
gram-negative Acceptance criterion : 104 CFU/g or CFU/mL are described under 5.1.1. Methods of preparation of sterile
bacteria (2.6.31) products. The method of media-ll runs may be used to
Escherichia coli evaluate the process of aseptic production. Apart from that,
Absence (1 g or 1 mL)
(2.6.31) the sterility test is the only analytical method available for
Salmonella (2.6.31) Absence (25 g or 25 mL) products prepared under aseptic conditions and furthermore
it is, in all cases, the only analytical method available to the
authorities who have to examine a specimen of a product for
EXTRACTS sterility.
Extracts should fulll the acceptance criteria for category B The probability of detecting micro-organisms by the test for
herbal medicinal products. However, where it can be sterility increases with their number present in the sample
demonstrated that the method of processing would not tested and varies according to the readiness of growth of
reduce the level of micro-organisms sufciently to reach the micro-organism present. The probability of detecting very
category B criteria, the extracts shall meet the requirements low levels of contamination even when it is homogenous
for category C herbal medicinal products. throughout the batch is very low. The interpretation of the
The recommended acceptance criteria apply to extracts that results of the test for sterility rests on the assumption that
are to be incorporated into herbal medicinal products for the contents of every container in the batch, had they been
oral use. More-stringent acceptance criteria may be required tested, would have given the same result. Since it is manifest
for extracts that are to be incorporated into pharmaceutical that every container cannot be tested, an appropriate sampling
preparations to be administered by other routes in order plan should be adopted. In the case of aseptic production, it is
to satisfy the acceptance criteria for the intended route of recommended to include samples lled at the beginning and
administration (5.1.4). at the end of the batch and after signicant intervention.
It is recognised that for some herbal medicinal products and OBSERVATION AND INTERPRETATION OF RESULTS
extracts used in their preparation the criteria given above for
TAMC, TYMC and bile-tolerant gram-negative bacteria cannot Conventional microbiological/biochemical techniques are
be met because of the typical level of microbial contamination. generally satisfactory for identication of micro-organisms
Less-stringent acceptance criteria may be applied on the basis recovered from a sterility test. However, if a manufacturer
of a risk assessment that takes account of qualitative and wishes to use condition (d) as the sole criterion for invalidating
quantitative characterisation of the microbial contamination a sterility test, it may be necessary to employ sensitive typing
and the intended use of the herbal medicinal product or extract. techniques to demonstrate that a micro-organism isolated
from the product test is identical to a micro-organism isolated
If it has been shown that none of the prescribed tests for a herbal
medicinal product or extract will allow valid enumeration of from the test materials and/or the testing environment. While
micro-organisms at the level prescribed, a validated method routine microbiological/biochemical identication techniques
with a limit of detection as close as possible to the indicated can demonstrate that 2 isolates are not identical, these
acceptance criterion is used. methods may not be sufciently sensitive or reliable enough to
provide unequivocal evidence that 2 isolates are from the same
source. More sensitive tests, for example molecular typing
01/2009:50109 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
01/2010:50110
The purpose of the test for sterility (2.6.1), as that of all
pharmacopoeial tests, is to provide an independent control
analyst with the means of verifying that a particular material 5.1.10. GUIDELINES FOR USING THE
meets the requirements of the European Pharmacopoeia. A TEST FOR BACTERIAL ENDOTOXINS
manufacturer is neither obliged to carry out such tests nor
precluded from using modications of, or alternatives to, the 1. INTRODUCTION
stated method, provided he is satised that, if tested by the Endotoxins from gram-negative bacteria are the most
ofcial method, the material in question would comply with common cause of toxic reactions resulting from contamination
the requirements of the European Pharmacopoeia. of pharmaceutical products with pyrogens ; their pyrogenic
activity is much higher than that of most other pyrogenic
PRECAUTIONS AGAINST MICROBIAL substances. These endotoxins are lipo-polysaccharides.
CONTAMINATION Although there are a small number of pyrogens which possess
Aseptic conditions for performance of the test can be achieved a different structure, the conclusion is generally justied that
using, for example, a class A laminar-air-ow cabinet located the absence of bacterial endotoxins in a product implies the
within a class B clean room, or an isolator absence of pyrogenic components, provided the presence of
non-endotoxin pyrogenic substances can be ruled out.
GUIDANCE TO MANUFACTURERS The presence of endotoxins in a product may be masked
The level of assurance provided by a satisfactory result of a by factors interfering with the reaction between the
test for sterility (the absence of contaminated units in the endotoxins and the amoebocyte lysate. Hence, the analyst
sample) as applied to the quality of the batch is a function of who wishes to replace the rabbit pyrogen test required in a
the homogeneity of the batch, the conditions of manufacture pharmacopoeial monograph by a test for bacterial endotoxins

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

EUROPEAN PHARMACOPOEIA 8.0
has to demonstrate that a valid test can be carried out on the
product concerned ; this may entail a procedure for removing
interfering factors.
As indicated in the test for bacterial endotoxins (2.6.14),
information must be available on the 2 following aspects
before a test on a sample can be regarded as valid.
The suitability of the material to be used for the test has
to be established. The absence of endotoxins in the water
for BET and in the other reagents must be assured and the when the endotoxin concentration is much higher, and the
sensitivity of the amoebocyte lysate must be checked to
conrm the sensitivity declared by the manufacturer.
As the product to be examined may interfere with the
test, the sensitivity of the amoebocyte lysate is determined and one that is higher. It is only when no gelation occurs that
in the presence and in the absence of the product under
examination. There must be no signicant difference
between the 2 sensitivity values.
The text 2.6.14. Bacterial endotoxins indicates methods for
removing interfering factors ; in the case of interference,
another test must be carried out after such a method has been per millilitre of solution to be tested, as the test can only be
applied to check whether the interference has indeed been
neutralised or removed.
This general chapter explains the reasons for the requirements
in the test for bacterial endotoxins, then deals with the reading
and interpretation of the results.
Substitution of the rabbit pyrogen test required in a
pharmacopoeial monograph by an amoebocyte lysate test
constitutes the use of an alternative method of analysis and
hence requires validation ; some guidance on how to proceed
is given in section 11.
The reference method for bacterial endotoxins is stated in the
monograph on a given product ; where no method is stated,
method A is the reference method. If a method other than the
reference method is to be used, the analyst must demonstrate
that the method is appropriate for this product and gives a
result consistent with that obtained with the reference method
(see also Section 13).
2. METHOD
The addition of endotoxins to amoebocyte lysate may result in For other routes, the acceptance criterion for bacterial
turbidity, precipitation or gelation (gel-clot) ; only the gel-clot endotoxins is generally determined on the basis of results
method was used in the Pharmacopoeia as an evaluation
criterion in the rst type of test for bacterial endotoxins. The
advantage was the simplicity of basing the decision to pass or
fail the product under examination on the absence or presence Route of administration
of a gel-clot, visible with the naked eye. The quantitative
methods described as methods C, D, E and F were developed Intravenous
later : they require more instrumentation, but they are easier
to automate for the regular testing of large numbers of samples
of the same product.
Endotoxins may be adsorbed onto the surface of tubes
or pipettes made from certain plastics or types of glass.
Interference may appear due to the release of substances
from plastic materials. Hence, the materials used should be
checked ; subsequent batches of tubes or pipettes may have
a slightly different composition, and therefore the analyst is
advised to repeat such tests on starting with new batches of
materials.
The decision to use the test for bacterial endotoxins as a limit
test implies rst that a threshold endotoxin concentration
must be dened for the product to be tested, and second that
the objective of the test is to know whether the endotoxin
concentration in the product under examination is below or
above this threshold. The quantitative methods C, D, E and F
make it possible to determine the endotoxin concentration
in the sample under examination, but for compliance with
the Pharmacopoeia and in routine quality control the nal
question is whether or not this concentration exceeds a
dened limit.
General Notices (1) apply to all monographs and other texts
5.1.10. Guidelines for using the test for bacterial endotoxins
In setting a threshold concentration of endotoxin for the
product to be tested, due attention should be paid to the dose
of the product : the threshold should be set so as to ensure that
as long as the endotoxin concentration in the product remains
below this threshold even the maximal dose administered
by the intended route per hour does not contain sufcient
endotoxin to cause a toxic reaction.
When the endotoxin concentration in the product exactly
equals the threshold value, gelation will occur, as is the case
product will fail the test, because the all-or-none character
of the test makes it impossible to differentiate between a
concentration exactly equal to the threshold concentration
the analyst may conclude that the endotoxin concentration is
below the threshold concentration.
For products in the solid state, this threshold concentration
of endotoxin per mass unit or per International Unit (IU) of
product has to be translated into a concentration of endotoxin
carried out on a solution. The case of products that already
exist in the liquid state (such as infusion uids) is discussed
below.
Endotoxin limit : the endotoxin limit for active substances
administered parenterally, dened on the basis of dose, is
equal to :
K = threshold pyrogenic dose of endotoxin per
kilogram of body mass ;
M = maximum recommended bolus dose of product
per kilogram of body mass.
When the product is to be injected at frequent intervals
or infused continuously, M is the maximum total dose
administered in a single hour period.
The endotoxin limit depends on the product and its route of
administration and is stated in the monograph. Values for K
are suggested in Table 5.1.10.-1.
obtained during the development of the preparation.
Table 5.1.10.-1
K (IU of endotoxin per kilogram
of body mass)
5.0
Intravenous, for radiopharmaceuticals 2.5
Intrathecal 0.2
Which dilution of the product is to be used in the test to
obtain maximal assurance that a negative result means that
the endotoxin concentration of the product is less than the
endotoxin limit and that a positive result means that the
lysate detected an endotoxin concentration equal to or greater
than the endotoxin limit? This dilution depends on the
endotoxin limit and on the sensitivity of the lysate : it is called
the Maximum Valid Dilution (MVD) and its value may be
calculated using the following expression :
Concentration of test solution :
mg/mL if the endotoxin limit is specied by mass (IU/mg) ;
Units/mL if the endotoxin limit is specied by unit of
biological activity (IU/Unit) ;
mL/mL if the endotoxin limit is specied by volume
(IU/mL).
573
5.1.10. Guidelines for using the test for bacterial endotoxins EUROPEAN PHARMACOPOEIA 8.0

= the labelled lysate sensitivity in the gel-clot technique analysts may prefer to distil the water more than 3 times.
(IU/mL) or the lowest concentration used in the Whatever method is used, the resultant product must be free
standard curve of the turbidimetric or chromogenic of detectable endotoxins.
techniques.
5. pH OF THE MIXTURE
When the value of the maximum valid dilution is not a whole
number, a convenient whole number smaller than the MVD In the test for bacterial endotoxins, optimum gel-clot occurs
may be used for routine purposes (which means preparing a for a mixture at pH 6.0-8.0. However, the addition of the lysate
solution of the product which is less diluted than the MVD to the sample may result in a lowering of the pH.
indicates). In this case, a negative result indicates that the
endotoxin concentration of the product lies below the limit 6. VALIDATION OF THE LYSATE
value. However, when the endotoxin concentration of the It is important to follow the manufacturers instructions for
product in such a test is less than the endotoxin limit but the preparation of the solutions of the lysate.
high enough to make the reaction with the lysate result in a
The positive end-point dilution factors in gel-clot methods A
clot, the test may be positive under these conditions. Hence,
and B are converted to logarithms. The reason is that if the
when a test with this convenient dilution factor is positive,
frequency distribution of these logarithmic values is plotted, it
the product should be diluted to the MVD and the test should
usually approaches a normal distribution curve much more
be repeated. In any case of doubt or dispute the MVD must
closely than the frequency distribution of the dilution factors
be used.
themselves ; in fact it is so similar that it is acceptable to use
This stresses the importance of the conrmation of the the normal frequency distribution as a mathematical model
sensitivity of the lysate. and to calculate condence limits with Students t-test.
Example
A 50 mg/mL solution of phenytoin sodium (intended for 7. PRELIMINARY TEST FOR INTERFERING FACTORS
intravenous injection) has to be tested. Determine the MVD, Some products cannot be tested directly for the presence of
given the following variables : endotoxins because they are not miscible with the reagents,
M = maximum human dose = 15 mg per kilogram of they cannot be adjusted to pH 6.0-8.0 or they inhibit or
body mass ; activate gel formation. Therefore a preliminary test is required
to check for the presence of interfering factors ; when these
c = 50 mg/mL ; are found the analyst must demonstrate that the procedure to
K = 5 IU of endotoxin per kilogram of body mass ; remove them has been effective.
= 0.4 IU of endotoxin per millilitre. The object of the preliminary test is to test the null hypothesis
that the sensitivity of the lysate in the presence of the product
under examination does not differ signicantly from the
sensitivity of the lysate in the absence of the product. A simple
criterion is used in methods A and B : the null hypothesis is
For routine tests on this product, it may be expedient to dilute accepted when the sensitivity of the lysate in the presence of
1 mL of the solution to be tested to 20 mL (MVD/2 rounded the product is at least 0.5 times and not more than twice the
to the next lower whole number). However, if this test result is sensitivity of the lysate by itself.
positive the analyst will have to dilute 1 mL to 41.67 mL and
A classical approach would have been to calculate the means
repeat the test. A dilution to 41.67 mL is also necessary when
of the log dilution factor for the lysate sensitivity with and
the test is performed to settle a dispute.
without the product and to test the difference between the
3. REFERENCE MATERIAL 2 means with Students t-test.
Endotoxin standard BRP is intended for use as the reference The test for interfering factors in gel-clot methods A and B
preparation. It has been assayed against the WHO requires the use of a sample of the product in which no
International Standard for Endotoxin and its potency is endotoxins are detectable. This presents a theoretical problem
expressed in International Units of endotoxin per ampoule. when an entirely new product has to be tested. Hence, a
The International Unit of endotoxin is dened as the specic different approach was designed for quantitative methods C,
activity of a dened mass of the International Standard. D, E and F.
For routine purposes, another preparation of endotoxin may
be used, provided it has been assayed against the International 8. REMOVAL OF INTERFERING FACTORS
Standard for Endotoxin or the BRP and its potency is The procedures to remove interfering factors must not
expressed in International Units of endotoxin. increase or decrease (for example, by adsorption) the amount
NOTE : 1 International Unit (IU) of endotoxin is equal to of endotoxin in the product under examination. The correct
1 Endotoxin Unit (E.U.). way of checking this is to apply the procedures to a spiked
sample of the product, that is, a sample to which a known
4. WATER FOR BET amount of endotoxin has been added, and then to measure
Testing the absence of endotoxin in this reagent by a technique the recovery of the endotoxin.
derived from the rabbit pyrogen test was rejected for practical Methods C and D. If the nature of the product to be analysed
and theoretical reasons : shows interference which cannot be removed by classical
the rabbit test is not sensitive enough to detect endotoxin methods, it may be possible to determine the standard curve in
in water for BET intended for tests on products with a very the same type of product freed from endotoxins by appropriate
low endotoxin limit ; treatment or by dilution of the product. The endotoxins test is
the relatively low precision of the rising temperature then carried out by comparison with this standard curve.
response in rabbits would call for many replications in Ultraltration with cellulose triacetate asymmetric membrane
rabbits ; lters has been found to be suitable in most cases. The
the terms pyrogens and endotoxins denote groups of lters should be properly validated, because under some
entities that do not coincide completely. circumstances cellulose derivatives (-D-glucans) can cause
The text 2.6.14. Bacterial endotoxins indicates that methods false positive results.
other than triple distillation may be used to prepare water for Polysulfone lters have been found to be unsuitable because
BET. Reverse osmosis has been used with good results ; some false positive results had been obtained by some users.

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

EUROPEAN PHARMACOPOEIA 8.0
9. THE PURPOSE OF THE CONTROLS
The purpose of the control made up with water for BET
and the reference preparation of endotoxin at twice the
concentration of the labelled lysate sensitivity is to verify the
activity of the lysate at the time and under the conditions of
the test. The purpose of the negative control is to verify the
absence of a detectable concentration of endotoxin in water
for BET.
The positive control, which contains the product to be
examined at the concentration used in the test, is intended to
show the absence of inhibiting factors at the time and under
the conditions of the test.
10. READING AND INTERPRETATION OF THE RESULTS
Minute amounts of endotoxin in the water for BET, or in
any other reagent or material to which the lysate is exposed
during the test, may escape detection as long as they do not
reach the sensitivity limit of the lysate. However, they may
raise the amount of endotoxin in the solution containing the
product under examination to just above the sensitivity limit
and cause a positive reaction.
The risk of this happening may be reduced by testing the
water for BET and the other reagents and materials with the
most sensitive lysate available, or at least one that is more
sensitive than the one used in the test on the product. Even
then, the risk of such a false positive result cannot be ruled
out completely. It should be realised, however, that in this
respect the test design is fail-safe in contrast to a test design
permitting a false negative result, which could lead to the
release of an unsatisfactory product, thus endangering the
patients health.
11. REPLACEMENT OF THE RABBIT PYROGEN TEST BY
A TEST FOR BACTERIAL ENDOTOXINS
Monographs on pharmaceutical products intended for
parenteral administration that may contain toxic amounts
of bacterial endotoxins require either a test for bacterial
endotoxins or a rabbit pyrogen test. As a general policy :
in any individual monograph, when a test is required, only
one test is included, either that for pyrogens or that for
bacterial endotoxins ;
in the absence of evidence to the contrary, the test for
bacterial endotoxins is preferred over the test for pyrogens,
since it is usually considered to provide equal or better
protection to the patient ;
before including a test for bacterial endotoxins in a
monograph, evidence is required that one of the tests
described in chapter 2.6.14 can be applied satisfactorily to
the product in question ;
the necessary information is sought from manufacturers ;
companies are invited to provide any validation data
that they have concerning the applicability of the test for
bacterial endotoxins to the substances and formulations of
General Notices (1) apply to all monographs and other texts
5.1.10. Guidelines for using the test for bacterial endotoxins
interest ; such data includes details of sample preparation
and of any procedures necessary to eliminate interfering
factors ; in addition, any available parallel data for rabbit
pyrogen testing that would contribute to an assurance that
the replacement of a rabbit pyrogen test by the test for
bacterial endotoxin is appropriate, must be provided.
Additional requirements are dened in the following sections.
12. USE OF A DIFFERENT BACTERIAL ENDOTOXIN
TEST FROM THAT PRESCRIBED IN THE MONOGRAPH
When a test for bacterial endotoxins is prescribed in a
monograph and none of the 6 methods (A to F) described in
chapter 2.6.14 is specied, then method A, the gel-clot method
limit test, has been validated for this product. If one of the
other methods (B to F) is specied, this is the one which has
been validated for this product.
13. VALIDATION OF ALTERNATIVE METHODS
Replacement of a rabbit pyrogen test by a bacterial endotoxin
test, or replacement of a stated or implied method for bacterial
endotoxins by another method, is to be regarded as the use of
an alternative method in the replacement of a pharmacopoeial
test, as described in the General Notices :
The test and assays described are the ofcial methods
upon which the standards of the Pharmacopoeia are based.
With the agreement of the competent authority, alternative
methods of analysis may be used for control purposes,
provided that the methods used enable an unequivocal
decision to be made as to whether compliance with the
standards of the monographs would be achieved if the
ofcial methods were used. In the event of doubt or
dispute, the methods of analysis of the Pharmacopoeia are
alone authoritative.
The following procedures are suggested for validating a
method for bacterial endotoxins other than the one implied or
indicated in the monograph.
13-1. The procedure and the materials and reagents used
in the method should be validated as described for the test
concerned.
13-2. The presence of interfering factors (and, if needed, the
procedure for removing them) should be tested on samples
of at least 3 production batches. It should be borne in mind
that methods D and E, using a chromogenic peptide, require
reagents that are absent in methods A, B, C and F, and hence
compliance of methods A, B, C or F with the requirements
for interfering factors cannot be extrapolated to method D or
method E without further testing.
14. VALIDATION OF THE TEST FOR NEW PRODUCTS
The procedures described under 13-1 and 13-2 should
be applied to all new products intended for parenteral
administration that have to be tested for the presence of
bacterial endotoxins according to the requirements of the
Pharmacopoeia.
575