EUROPEAN PHARMACOPOEIA 9.0 5.1.10.

Guidelines for using the test for bacterial endotoxins

methods may not be sufficiently sensitive or reliable enough to test using recombinant factor C reagent as a replacement for
provide unequivocal evidence that 2 isolates are from the same the amoebocyte lysate, constitutes the use of an alternative
source. More sensitive tests, for example molecular typing method of analysis and hence requires demonstration that
with RNA/DNA homology, may be necessary to determine the method is appropriate for the given substance or product
that micro-organisms are clonally related and have a common and gives a result consistent with that obtained with the
origin. prescribed method as described in the General Notices (see
also section 12).
The prescribed method for bacterial endotoxins may be stated
in the monograph on a given substance or product. The use of
07/2016:50110 a method other than the method prescribed in the monograph
is considered as the use of an alternative method. Where no
method is stated, any of methods A to F of general chapter
2.6.14. Bacterial endotoxins can be used.

5.1.10. GUIDELINES FOR USING THE 2. METHOD AND ACCEPTANCE CRITERIA

TEST FOR BACTERIAL ENDOTOXINS 2-1. METHODS AND PRECAUTIONS TO BE TAKEN
The addition of endotoxins to amoebocyte lysate may result
1. INTRODUCTION in turbidity, precipitation or gelation (gel-clot) ; initially only
Endotoxins from gram-negative bacteria are the the gel-clot method was used in the Pharmacopoeia as an
most common cause of toxic reactions resulting from evaluation criterion in the test for bacterial endotoxins. The
contamination of pharmaceutical products with pyrogens ; advantage was the simplicity of basing the decision to pass or
their common pyrogenic activity is much higher than that fail the substance or product to be examined on the absence
of other known pyrogenic substances. These endotoxins or presence of a gel-clot, visible with the naked eye. The
are lipopolysaccharides. Although there are a small quantitative methods C, D, E and F were developed later : they
number of pyrogens that possess a different structure, require more instrumentation, but they are easier to automate
the conclusion is generally justified that the absence of for the regular testing of large numbers of samples of the same
bacterial endotoxins in a substance or product implies the substance or product.
absence of pyrogenic components, provided the presence of
non-endotoxin pyrogenic substances can be ruled out. The Endotoxins may be adsorbed onto the surface of tubes
monocyte-activation test (2.6.30) is a suitable method to or pipettes made from certain plastics or types of glass.
use to rule out the presence of non-endotoxin pyrogens in Interference may appear due to the release of substances from
substances or products. plastic materials. Hence, the materials used must be checked.
The presence of endotoxins in a substance or product may 2-2. ENDOTOXIN LIMIT CONCENTRATION
be masked by factors interfering with the reaction between The decision to use the test for bacterial endotoxins as a limit
the endotoxins, the test reagents and the amoebocyte lysate. test implies firstly that an endotoxin limit concentration must
Also, the ability to detect endotoxins may be affected by be defined for the substance or product to be examined, and
storage conditions or storage time. Hence, the analyst who secondly that the objective of the test is to know whether the
wishes to implement a test for bacterial endotoxins or to endotoxin concentration in the sample to be examined is below
replace the pyrogen test by a test for bacterial endotoxins has or above this limit. The quantitative methods C, D, E and F
to demonstrate that a valid test can be carried out on the make it possible to determine the endotoxin concentration
substance or product concerned ; this may entail a procedure in the sample to be examined, but for compliance with the
for removing interference. Pharmacopoeia and in routine quality control the final
As indicated in general chapter 2.6.14. Bacterial endotoxins, question is whether or not this concentration exceeds a
information must be available on the following 2 aspects defined limit.
before a test on a sample can be regarded as valid.
The dose of the substance or product to be examined
– The suitability of the material to be used for the test has must be taken into account in setting the endotoxin limit
to be established. The absence of endotoxins in the water concentration : the limit is set so as to ensure that, as long
for BET (water for bacterial endotoxins test) and in the as the endotoxin concentration in the substance or product
other reagents and consumables must be assured and the remains below this limit, even the maximal dose administered
sensitivity of the amoebocyte lysate must be checked to by the intended route per hour does not contain sufficient
confirm the sensitivity declared by the manufacturer. endotoxin to cause a toxic reaction.
– As the substance or product to be examined may interfere
When the endotoxin concentration in the substance or product
with the test, the sensitivity of the amoebocyte lysate is
exactly equals the endotoxin limit concentration, gelation will
determined in the presence and in the absence of the
occur, as is the case when the endotoxin concentration is much
substance or product to be examined. There must be no
higher, and the substance or product will fail the test, because
difference between the 2 sensitivity values.
the all-or-none character of the test makes it impossible to
General chapter 2.6.14. Bacterial endotoxins indicates differentiate between a concentration exactly equal to the
methods for removing interfering factors ; in the case of endotoxin limit concentration and one that is higher. It is only
interference, another test must be carried out after such a when no gelation occurs that the analyst may conclude that
method has been applied to check whether the interference the endotoxin concentration is below the endotoxin limit.
has indeed been neutralised or removed.
For substances or products in the solid state, this endotoxin
This general chapter explains the reasons for the requirements limit concentration per mass unit or per International Unit
in the test for bacterial endotoxins, then deals with reading (IU) of substance or product has to be converted into a
and interpretation of the results. concentration of endotoxin per millilitre of solution to be
Replacement of the rabbit pyrogen test required in a examined, as the test can only be carried out on a solution.
pharmacopoeial monograph by an amoebocyte lysate test, or The case of substances or products that already exist in the
by other methods such as the monocyte-activation test or a liquid state (such as infusion fluids) is discussed below.

General Notices (1) apply to all monographs and other texts 593
5.1.10. Guidelines for using the test for bacterial endotoxins EUROPEAN PHARMACOPOEIA 9.0

2-3. CALCULATION OF THE ENDOTOXIN LIMIT the endotoxin limit and on the sensitivity of the lysate ; it is
The endotoxin limit for active substances administered called the maximum valid dilution (MVD) and its value may
parenterally, defined on the basis of dose, is equal to : be calculated using the following expression :

K = threshold pyrogenic dose of endotoxin per Concentration of test solution :

kilogram of body mass ; – in mg/mL if the endotoxin limit is specified by mass
M = maximum recommended bolus dose of product (IU/mg) ;
per kilogram of body mass. – in Units/mL if the endotoxin limit is specified by unit of
When the product is to be injected at frequent intervals biological activity (IU/Unit) ;
or infused continuously, M is the maximum total dose – in mL/mL if the endotoxin limit is specified by volume
administered per hour. (IU/mL).
The endotoxin limit depends on the product and its route of λ = the labelled lysate sensitivity in the gel-clot technique
administration and may be stated in the monograph. Values (IU/mL) or the lowest concentration used in the
for K are suggested in Table 5.1.10.-1. standard curve of the turbidimetric or chromogenic
For other routes, the acceptance criterion for bacterial techniques.
endotoxins is generally determined on the basis of results When the value of the MVD is not a whole number, a
obtained during the development of the preparation. convenient whole number smaller than the MVD may be
Table 5.1.10.-1 used for routine purposes (which means preparing a solution
of the substance or product that is less diluted than the
Route of administration K
MVD indicates). In this case, a negative result indicates that
Intravenous 5.0 IU of endotoxin per kilogram the endotoxin concentration of the substance or product
of body mass lies below the limit value. However, when the endotoxin
Intravenous for radiopharmaceuticals 2.5 IU of endotoxin per kilogram concentration of the substance or product in such a test is less
of body mass than the endotoxin limit but high enough to make the reaction
Intrathecal 0.2 IU of endotoxin per kilogram
with the lysate result in a clot, the test may be positive under
of body mass these conditions. Hence, when a test with this ‘convenient’
2
dilution factor is positive, the substance or product is diluted
Parenteral formulations administered 100 IU/m
per square metre of body surface to the MVD and the test is repeated. In any case of doubt or
dispute, the MVD must be used.
2-4. CONSIDERATIONS WHEN ESTABLISHING AN This stresses the importance of the confirmation of the
ENDOTOXIN LIMIT FOR A SPECIFIC SUBSTANCE OR sensitivity of the lysate.
PRODUCT
Example
The endotoxin limit for a substance or product is established
with consideration of the following aspects. A 50 mg/mL solution of phenytoin sodium (intended for
intravenous injection) has to be tested. Determine the MVD,
Calculated endotoxin limit. The endotoxin limit is calculated
given the following variables :
as described in section 2-3. This represents a safety limit not
to be exceeded if the product is to be administered to humans. M = maximum human dose = 15 mg per kilogram of
body mass ;
Limit prescribed in an individual substance monograph. c = 50 mg/mL ;
The limit stated in an individual substance monograph
frequently reflects what is achievable in a controlled K = 5 IU of endotoxin per kilogram of body mass ;
production environment. The limit prescribed in a = 0.4 IU of endotoxin per millilitre.
λ
monograph can therefore be lower than the calculated
endotoxin limit. However a manufacturer may specify a limit
that is more stringent than that stated in the monograph.
Process capability. The capability of the process to reduce or
remove bacterial endotoxins during manufacture might result For routine tests on this product, it may be expedient to
in lower endotoxin limits for specific processes. dilute 1 mL of the solution to be examined to 20 mL (MVD/2
Additional safety requirements. Precautions are taken rounded to the next lower whole number). However, if this
in consideration of patient population (such as paediatric test result is positive the analyst will have to dilute 1 mL to
use, malnourished or cachectic patients, etc.), specific local 41.67 mL and repeat the test. A dilution to 41.67 mL is also
requirements (e.g. countries might wish to operate with a necessary when the test is performed to settle a dispute.
lower average body weight of 60 kg instead of 70 kg frequently
employed in Europe) or any additional safety margins 3. RISK ASSESSMENT
requested by the competent authority. As stated in section 1 of this general chapter, the conclusion
is generally justified that the absence of bacterial endotoxins
Formulation of the product. The limit must take into
in a substance or product implies the absence of pyrogenic
consideration any theoretical bacterial endotoxin load
components, provided the presence of non-endotoxin
introduced by any other components used for reconstitution
pyrogenic substances can be ruled out. To rule out the
and/or dilution of the product (e.g. water for injections) or
presence of non-endotoxin pyrogens in substances or
introduced by starting materials and/or raw materials.
products, the use of the monocyte-activation test (2.6.30)
2-5. MAXIMUM VALID DILUTION is recommended at release or during development of
Which dilution of the substance or product is to be used in the the production process ; if any changes are made to the
test to obtain maximal assurance that a negative result means production process that could influence the quality of the
that the endotoxin concentration of the substance or product product regarding pyrogenicity, the monocyte-activation
is less than the endotoxin limit and that a positive result means test is repeated. Examples of such changes include the use
that the lysate detected an endotoxin concentration equal to of different raw materials, a different production site and
or greater than the endotoxin limit? This dilution depends on different process parameters.

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

EUROPEAN PHARMACOPOEIA 9.0 5.1.10. Guidelines for using the test for bacterial endotoxins

The decision to use the test for bacterial endotoxins as the sole usually approaches a normal distribution curve much more
pyrogenicity test is to be made after careful evaluation of the closely than the frequency distribution of the dilution factors
risk of the substance or product containing non-endotoxin themselves ; in fact it is so similar that it is acceptable to use
pyrogens. The risk assessment is made with consideration the normal frequency distribution as a mathematical model
given to any factor that could result in the inclusion of and to calculate confidence limits with Student’s t-test.
pyrogens not detected by the test for bacterial endotoxins. The
items below constitute a non-exhaustive list of factors to be 8. PRELIMINARY TEST FOR INTERFERING FACTORS
considered in the risk assessment. Some substances or products cannot be tested directly for the
Production process (chemical synthesis, fermentation, presence of bacterial endotoxins because they are not miscible
biotechnological method). For products of fermentation, with the reagents, they cannot be adjusted to pH 6.0-8.0 or they
the expression system is to be considered (prokaryotic, inhibit or activate enzymatic reaction (such as β-D-glucans).
eukaryotic) and, for a prokaryotic expression system, whether Therefore a preliminary test is required to check for the
gram-positive or gram-negative bacteria are used. Also, the presence of interfering factors ; when these are found the
culture media components are examined with consideration analyst must demonstrate that the procedure to remove them
given to their origin (synthetic, animal, plant). has been effective and that by applying this procedure, any
Bioburden. The potential presence of gram-positive bacteria bacterial endotoxins present have not been removed.
and fungi as contaminants of the active substance, excipients The object of the preliminary test is to test the null hypothesis
or starting materials and raw materials used in the production that the sensitivity of the lysate in the presence of the substance
of the medicinal product, and the origin of the raw materials or product to be examined does not differ significantly from
(synthetic, animal, plant) have to be taken into consideration. the sensitivity of the lysate in the absence of the product.
The quality of the water plays an important role on the overall A simple criterion is used in methods A and B : the null
evaluation. hypothesis is accepted when the sensitivity of the lysate in the
Capability of the downstream process. It must be verified presence of the product is at least 0.5 times and not more than
whether bacterial endotoxin removal steps are part of the twice the sensitivity of the lysate by itself.
downstream process. The test for interfering factors in gel-clot methods A and B
requires the use of a sample of the substance or product in
Safety. The target population and the route of administration which no endotoxins are detectable. This presents a theoretical
(e.g. intravenous, intrathecal) have to be taken into account problem when an entirely new product has to be tested.
in the risk assessment. Hence, a different approach was designed for quantitative
Stability of the detectable endotoxins. It has to be considered methods C, D, E and F.
that the ability to detect endotoxins can be affected by Note that methods D and E, which use a chromogenic peptide,
interaction with certain components, storage conditions or require reagents that are absent in methods A, B, C and F,
storage time, temperature and handling of the test sample. and hence compliance of methods A, B, C or F with the
Procedures that demonstrate stability of the detectable requirements for interfering factors cannot be extrapolated to
endotoxin content have to be established for storing, handling method D or method E without further testing.
and mixing of samples.
9. REMOVAL OF INTERFERING FACTORS
4. REFERENCE MATERIAL
Endotoxin standard BRP is intended for use as the reference The procedures to remove interfering factors must not
preparation. It has been assayed against the WHO increase or decrease (for example, by adsorption) the amount
International Standard for Endotoxin and its potency is of endotoxin in the substance or product to be examined. The
expressed in International Units of endotoxin per vial. The correct way of checking this is to apply the procedures to a
International Unit of endotoxin is defined as the specific spiked sample of the substance or product to be examined,
activity of a defined mass of the International Standard. that is, a sample to which a known amount of endotoxin
has been added, and then to measure the recovery of the
For routine purposes, another preparation of endotoxin may endotoxin after the removal process has been conducted.
be used, provided it has been assayed against the International
Standard for Endotoxin or the BRP and its potency is Methods C and D. If the nature of the product to be examined
expressed in International Units of endotoxin. results in an interference that cannot be removed by classical
methods (e.g. dilution or centrifugation), it may be possible to
NOTE : 1 International Unit (IU) of endotoxin is equal to
determine the standard curve in the same type of substance or
1 Endotoxin Unit (E.U.).
product freed from endotoxins by appropriate treatment or by
5. WATER FOR BET dilution of the substance or product. The endotoxins test is
Water for BET is sterile water that is free of detectable levels of then carried out by comparison with this standard curve.
endotoxin. Usually it is commercially available and certified. Ultrafiltration with cellulose triacetate asymmetric membrane
General chapter 2.6.14. Bacterial endotoxins indicates that filters has been found to be suitable in most cases. The filters
methods other than triple distillation may be used to prepare must be properly validated, because under some circumstances
water for BET. Reverse osmosis has been used with good cellulose derivatives (β-D-glucans) can cause false positive
results ; some analysts may prefer to distil the water more than results.
3 times. Whatever method is used, the resultant product must Another option to remove interfering factors is a 2-step
be free of detectable bacterial endotoxins. procedure in which 1) endotoxin within the interfering
sample is fixed on a solid phase, and 2) after removal of the
6. pH OF THE MIXTURE interfering substance (e.g. by washing) the endotoxin is
In the test for bacterial endotoxins, optimum gel-clot occurs detected unimpaired under suitable testing conditions.
for a mixture at pH 6.0-8.0. However, the addition of the lysate
to the sample may result in a lowering of the pH. 10. THE PURPOSE OF THE CONTROLS
The purpose of the control made up with water for BET
7. VALIDATION OF THE LYSATE and the reference preparation of endotoxin at twice the
It is important to follow the manufacturer’s instructions for concentration of the labelled lysate sensitivity is to verify the
the preparation of the solutions of the lysate. activity of the lysate at the time and under the conditions of
The positive end-point dilution factors in gel-clot methods A the test (for method A and B). The purpose of the negative
and B are converted to logarithms. The reason is that if the control is to verify the absence of a detectable concentration
frequency distribution of these logarithmic values is plotted, it of endotoxin in the water for BET.

General Notices (1) apply to all monographs and other texts 595
5.1.10. Guidelines for using the test for bacterial endotoxins EUROPEAN PHARMACOPOEIA 9.0

The positive control, which contains the product to be The procedure and the materials and reagents used in the
examined at the concentration used in the test, is intended to method must be validated as described for the test concerned.
show the absence of inhibiting factors at the time and under
the conditions of the test. The absence of interfering factors (and, if necessary, the
procedure for removing them) is verified on samples of at least
11. READING AND INTERPRETATION OF RESULTS 3 production batches.
Minute amounts of bacterial endotoxin in the water for BET,
or in any other reagent or material to which the lysate is The necessary information is sought from manufacturers ;
exposed during the test, may escape detection as long as they companies are invited to provide any validation data that they
do not reach the sensitivity limit of the lysate. However, they have concerning the applicability of the replacement test to
may raise the amount of bacterial endotoxin in the solution the substances and products of interest ; such data includes
containing the substance or product to be examined to just details of sample preparation and of any procedures necessary
above the sensitivity limit and cause a positive reaction. to eliminate interfering factors.
The risk of this happening may be reduced by testing the
water for BET and the other reagents and materials with the As stated in general chapter 2.6.30. Monocyte-activation
most sensitive lysate available, or at least one that is more test, the monocyte-activation test is primarily intended as
sensitive than the one used in the test on the product. Even a replacement of the rabbit pyrogen test. Guidelines on
then, the risk of such a ‘false positive result’ cannot be ruled which methods to use (A, B or C) and on how to validate
out completely. the monocyte-activation test are described in general chapter
2.6.30. Monocyte-activation test.
12. REPLACEMENT OF METHODS PRESCRIBED IN 12-2. REPLACEMENT BY AN ALTERNATIVE METHOD
MONOGRAPHS NOT DESCRIBED IN THE PH. EUR.
12-1. REPLACEMENT BY ANOTHER PH. EUR. METHOD The use of alternative reagents such as recombinant factor C
As stated in the General Notices, the test methods given in as a replacement to the amoebocyte lysate eliminates the use
monographs and general chapters have been validated in of a reagent extracted from live animals.
accordance with accepted scientific practice and current
recommendations on analytical validation. The methods Replacement of a rabbit pyrogen test or a bacterial endotoxin
described in general chapters 2.6.14. Bacterial endotoxins test prescribed in a monograph by a test using recombinant
and 2.6.30. Monocyte-activation test therefore do not have factor C reagent or any other reagent as a replacement of the
to be re-validated per se, other than in consideration of their amoebocyte lysate is to be regarded as the use of an alternative
use for a specific substance or product in a specific analytical method in the replacement of a pharmacopoeial test, as
environment. described in the General Notices.

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