PYROGEN/ENDOTOXIN TEST

PYROGEN: are products of metabolism of

microorganisms. The most potent pyrogenic substances
(endotoxins) are constituents (lipopolysaccharides, LPS)
of the cell wall of gram negative bacteria (eg,
Pseudomonas sp, Salmonella sp, Escherichia
coli). Gram-positive bacteria and fungi also produce
pyrogens but of lower potency and of different chemical
nature. Gram-positive bacteria produce peptidoglycans
wherease fungi product _-glucans, both of which can
cause non-endotoxin pyrogenic responses. Endotoxins
are lipopolysaccharides that typically exist in high
molecular weight aggregate forms. However,the
monomer unit of LPS is less than 10,000 daltons,
enabling endotoxin easily to pass through sterilizing 0.2
micron filters. Studies have shown that the lipid portion of
the molecule is responsible for the biological activity.
Since endotoxins are the most potent pyrogens and
gram-negative bacteria are ubiquitous in the environment,
especially water, this discussion focuses on endotoxins
and the risk of their presence as contaminants in sterile
products. Pyrogens, when present in parenteral drug
products and injected into patients, can cause fever,
chills, pain in the back and legs, and malaise. Although
pyrogenic reactions are rarely fatal, they can cause
serious discomfort and, in the seriously ill patient, shocklike symptoms that can be fatal. The intensity of the
pyrogenic response and its degree of hazard will be
affected by the medical condition of the patient, the
potency of the pyrogen, the amount of the pyrogen, and
the route of administration (intrathecal is most hazardous
followed by intravenous, intramuscular, and
subcutaneous). When bacterial (exogenous) pyrogens
are introduced into the body, LPS targets circulating
mononuclear cells (monocytes and macrophages) that, in
turn, produce pro-inflammatory cytokines such as
interleukin-2, interleukin-6, and tissue necrosis factor.
Besides LPS, gram-negative bacteria also release
many peptides (eg, exotoxin A, peptidoglycan, and
muramuyl peptides) that can mimic the activity of LPS
and induce cytokine release. The Limulus Amebocyte
Lysate (LAL) test, discussed later, can only detect the
presence of LPS. It has been suggested that a new test,
called Monocyte Activation Test, replace LAL as the
official pyrogen test because of its greater sensitivity to all
agents that induce the release of cytokines that cause
fever and a potential cascade of other adverse
physiological effects.

PYROGEN TEST
The pyrogen test is designed to limit to an acceptable
level the risks of febrile reaction in the patient to the
administration, by injection, of the product concerned.
The test involves measuring the rise in temperature of
rabbits following the intravenous injection of a test
solution and is designed for products that can be
tolerated by the test rabbit in a dose not to exceed 10 mL
per kg injected intravenously within a period of not more
than 10 minutes. For products that require preliminary
preparation or are subject to special conditions of
administration, follow the additional directions given in the
individual monograph or, in the case of antibiotics or
biologics, the additional directions given in the federal
regulations (see Biologics 1041 ).
APPARATUS AND DILUENTS
Render the syringes, needles, and glassware free from
pyrogens by heating at 250 for not less than 30 minutes
or by any other suitable method. Treat all diluents and
solutions for washing and rinsing of devices or parenteral
injection assemblies in a manner that will assure that they
are sterile and pyrogen-free. Periodically perform control
pyrogen tests on representative portions of the diluents
and solutions for washing or rinsing of the apparatus.
Where Sodium Chloride Injection is specified as a diluent,
use Injection containing 0.9 percent of NaCl.
TEMPERATURE RECORDING
Use an accurate temperature-sensing device such as a
clinical thermometer, or thermistor probes or similar
probes that have been calibrated to assure an accuracy
of 0.1 and have been tested to determine that a
maximum reading is reached in less than 5 minutes.
Insert the temperature-sensing probe into the rectum of
the test rabbit to a depth of not less than 7.5 cm, and,
after a period of time not less than that previously
determined as sufficient, record the rabbit's body
temperature.
TEST ANIMALS
Use healthy, mature rabbits. House the rabbits
individually in an area of uniform temperature between
20and 23 and free from disturbances likely to excite

them. The temperature varies not more than 3 from the

selected temperature. Before using a rabbit for the first
time in a pyrogen test, condition it not more than seven
days before use by a sham test that includes all of the
steps as directed for Procedure except injection. Do not
use a rabbit for pyrogen testing more frequently than
once every 48 hours, nor prior to 2 weeks following a
maximum rise of its temperature of 0.6 or more while
being subjected to the pyrogen test, or following its
having been given a test specimen that was adjudged
pyrogenic.
PROCEDURE
Perform the test in a separate area designated solely for
pyrogen testing and under environmental conditions
similar to those under which the animals are housed and
free from disturbances likely to excite them. Withhold all
food from the rabbits used during the period of the test.
Access to water is allowed at all times, but may be
restricted during the test. If rectal temperature-measuring
probes remain inserted throughout the testing period,
restrain the rabbits with light-fitting neck stocks that allow
the rabbits to assume a natural resting posture. Not more
than 30 minutes prior to the injection of the test dose,
determine the control temperature of each rabbit: this is
the base for the determination of any temperature
increase resulting from the injection of a test solution. In
any one group of test rabbits, use only those rabbits
whose control temperatures do not vary by more than 1
from each other, and do not use any rabbit having a
temperature exceeding 39.8 .
Unless otherwise specified in the individual monograph,
inject into an ear vein of each of three rabbits 10 mL of
the test solution per kg of body weight, completing each
injection within 10 minutes after start of administration.
The test solution is either the product, constituted if
necessary as directed in the labeling, or the material
under test treated as directed in the individual monograph
and injected in the dose specified therein. For pyrogen
testing of devices or injection assemblies, use washings
or rinsings of the surfaces that come in contact with the
parenterally administered material or with the injection
site or internal tissues of the patient. Assure that all test
solutions are protected from contamination. Perform the
injection after warming the test solution to a temperature
of 37 2 . Record the temperature at 30-minute intervals
between 1 and 3 hours subsequent to the injection.

TEST INTERPRETATION AND CONTINUATION

Consider any temperature decreases as zero rise. If no
rabbit shows an individual rise in temperature of 0.5 or
more above its respective control temperature, the
product meets the requirements for the absence of
pyrogens. If any rabbit shows an individual temperature
rise of 0.5 or more, continue the test using five other
rabbits. If not more than three of the eight rabbits show
individual rises in temperature of 0.5 or more and if the
sum of the eight individual maximum temperature rises
does not exceed 3.3, the material under examination
meets the requirements for the absence of pyrogens.
85 BACTERIAL ENDOTOXINS TEST
Portions of this general chapter have been harmonized
with the corresponding texts of the European
Pharmacopeia and/or the Japanese Pharmacopeia.
Those portions that are not harmonized are marked with
symbols (
) to specify this fact.
This chapter provides a test to detect or quantify bacterial
endotoxins that may be present in or on the sample of the
article(s) to which the test is applied. It uses Limulus
Amebocyte Lysate (LAL) obtained from the aqueous
extracts of circulating amebocytes of horseshoe crab
(Limulus polyphemus or Tachypleus tridentatus) which
has been prepared and characterized for use as an LAL
Reagent. 1
There are two types of techniques for this test: the gelclot techniques, which are based on gel formation, and
the photometric techniques. The latter include a
turbidimetric method, which is based on the development
of turbidity after cleavage of an endogenous substrate,
and a chromogenic method, which is based on the
development of color after cleavage of a synthetic
peptide-chromogen complex. Proceed by any one of
these techniques, unless otherwise indicated in the
monograph. In case of dispute, the final decision is based
on the gel-clot techniques, unless otherwise indicated in
the monograph.
In the gel-clot techniques, the reaction endpoint is
determined from dilutions of the material under test in
direct comparison with parallel dilutions of a reference
endotoxin, and quantities of endotoxin are expressed in
USP Endotoxin Units (USP-EU). [NOTEOne USP-EU is
equal to one IU of endotoxin.]

Because LAL Reagents have been formulated to be used

also for turbidimetric or colorimetric tests, such tests may
be used to comply with the requirements. These tests
require the establishment of a standard regression curve;
the endotoxin content of the test material is determined
by interpolation from the curve. The procedures include
incubation for a preselected time of reacting endotoxin
and control solutions with LAL Reagent and reading of
the spectrophotometric light absorbance at suitable
wavelengths. In the endpoint turbidimetric procedure the
reading is made immediately at the end of the incubation
period. In the endpoint colorimetric procedure the
reaction is arrested at the end of the preselected time by
the addition of an enzyme reaction-terminating agent
prior to the readings. In the turbidimetric and colorimetric
kinetic assays the absorbance is measured throughout
the reaction period and rate values are determined from
those readings.

Use an LAL Reagent of confirmed label sensitivity.

The validity of test results for bacterial endotoxins
requires an adequate demonstration that specimens of
the article or of solutions, washings, or extracts thereof to
which the test is to be applied do not of themselves inhibit
or enhance the reaction or otherwise interfere with the
test. Validation is accomplished by performing the
inhibition or enhancement test described under each of
the three techniques indicated. Appropriate negative
controls are included. Validation must be repeated if the
LAL Reagent source or the method of manufacture or
formulation of the article is changed.
Preparation of Sample Solutions

Depyrogenate all glassware and other heat-stable

materials in a hot-air oven using a validated process. 2
Commonly used minimum time and temperature
settings are 30 minutes at 250 . If employing plastic
apparatus, such as microplates and pipet tips for
automatic pipetters, use only that which has been shown
to be free of detectable endotoxin and not to interfere with
the test. [NOTEIn this chapter, the term tube includes
any other receptacle such as a micro-titer well.]

Prepare sample solutions by dissolving or diluting drugs

or extracting medical devices using LAL Reagent Water.
Some substances or preparations may be more
appropriately dissolved, diluted, or extracted in other
aqueous solutions. If necessary, adjust the pH of the
solution (or dilution thereof) to be examined so that the
pH of the mixture of the LAL Reagent and sample falls
within the pH range specified by the LAL Reagent
manufacturer. This usually applies to a product with a pH
in the range of 6.0 to 8.0. The pH may be adjusted using
an acid, base, or suitable buffer as recommended by the
LAL Reagent manufacturer. Acids and bases may be
prepared from concentrates or solids with LAL Reagent
Water in containers free of detectable endotoxin. Buffers
must be validated to be free of detectable endotoxin and
interfering factors.

PREPARATION OF THE STANDARD ENDOTOXIN

STOCK SOLUTION AND STANDARD SOLUTIONS

DETERMINATION OF MAXIMUM VALID DILUTION

(MVD)

The USP Endotoxin RS has a defined potency of 10,000

USP Endotoxin Units (EU) per vial. Constitute the entire
contents of 1 vial of the RSE with 5 mL of LAL Reagent
Water 3 , mix intermittently for 30 minutes, using a
vortex mixer, and use this concentrate for making
appropriate serial dilutions. Preserve the concentrate in a
refrigerator for making subsequent dilutions for not more
than 14 days. Mix vigorously, using a vortex mixer, for not
less than 3 minutes before use. Mix each dilution for not
less than 30 seconds before proceeding to make the next
dilution. Do not store dilutions, because of loss of activity
by adsorption, in the absence of supporting data to the
contrary.

The Maximum Valid Dilution is the maximum allowable

dilution of a specimen at which the endotoxin limit can be
determined. It applies to injections or to solutions for
parenteral administration in the form constituted or diluted
for administration, or, where applicable, to the amount of
drug by weight if the volume of the dosage form for
administration could be varied. The general equation to
determine MVD is:

APPARATUS AND GLASSWARE

Preparatory Testing

MVD = (Endotoxin limit Concentration of sample

solution) / ( )
where the concentration of sample solution and are as
defined below. Where the endotoxin limit concentration is
specified in the individual monograph in terms of volume
(in EU per mL), divide the limit by , which is the labeled

sensitivity (in EU per mL) of the LAL Reagent, to obtain

the MVD factor. Where the endotoxin limit concentration
is specified in the individual monograph in terms of weight
or Units of active drug (in EU per mg or in EU per Unit),
multiply the limit by the concentration (in mg per mL or in
Units per mL) of the drug in the solution tested or of the
drug constituted according to the label instructions,
whichever is applicable, and divide the product of the
multiplication by , to obtain the MVD factor. The MVD
factor so obtained is the limit dilution factor for the
preparation for the test to be valid.
ESTABLISHMENT OF ENDOTOXIN LIMITS
The endotoxin limit for parenteral drugs, defined on the
basis of dose, is equal to K/M, 4 where K is the
threshold human pyrogenic dose of endotoxin per kg of
body weight, and M is equal to the maximum
recommended human dose of product per kg of body
weight in a single hour period.
The endotoxin limit for parenteral drugs is specified in
individual monographs in units such as EU/mL, EU/mg, or
EU/Unit of biological activity.
GEL-CLOT TECHNIQUES
The gel-clot techniques detect or quantify endotoxins
based on clotting of the LAL Reagent in the presence of
endotoxin. The concentration of endotoxin required to
cause the lysate to clot under standard conditions is the
labeled sensitivity of the LAL Reagent. To ensure both the
precision and validity of the test, tests for confirming the
labeled LAL Reagent sensitivity and for interfering factors
are described under Preparatory Testing for the Gel-Clot
Techniques.
Preparatory Testing for the Gel-Clot Techniques
Test for Confirmation of Labeled LAL Reagent Sensitivity
Confirm the labeled sensitivity using at least 1 vial of
the LAL Reagent lot. Prepare a series of two-fold dilutions
of the USP Endotoxin RS in LAL Reagent Water to give
concentrations of 2 , , 0.5 , and 0.25 , where is as
defined above. Perform the test on the four standard
concentrations in quadruplicate and include negative
controls. The test for confirmation of lysate sensitivity is to
be carried out when a new batch of LAL Reagent is used
or when there is any change in the experimental
conditions that may affect the outcome of the test.

Mix a volume of the LAL Reagent with an equal volume

(such as 0.1-mL aliquots) of one of the standard solutions
in each test tube. When single test vials or ampuls
containing lyophilized LAL Reagent are used, add
solutions directly to the vial or ampul. Incubate the
reaction mixture for a constant period according to
directions of the LAL Reagent manufacturer (usually at 37
1 for 60 2 minutes), avoiding vibration. To test the
integrity of the gel, take each tube in turn directly from the
incubator and invert it through about 180 in one smooth
motion. If a firm gel has formed that remains in place
upon inversion, record the result as positive. A result is
negative if an intact gel is not formed. The test is not valid
unless the lowest concentration of the standard solutions
shows a negative result in all replicate tests.
The endpoint is the last positive test in the series of
decreasing concentrations of endotoxin. Calculate the
mean value of the logarithms of the endpoint
concentration and then the antilogarithm of the mean
value using the following equation:
Geometric Mean Endpoint Concentration = antilog (e /
f),
where e is the sum of the log endpoint concentrations
of the dilution series used, and f is the number of
replicate test tubes. The geometric mean endpoint
concentration is the measured sensitivity of the LAL
Reagent (in EU/mL). If this is not less than 0.5 and not
more than 2 , the labeled sensitivity is confirmed and is
used in tests performed with this lysate.
Interfering Factors Test for the Gel-Clot Techniques
Prepare solutions A, B, C, and D as shown in Table 1,
and perform the inhibition/enhancement test on the
sample solutions at a dilution less than the MVD, not
containing any detectable endotoxins, following the
procedure in the Test for Confirmation of Labeled LAL
Reagent Sensitivity above. The geometric mean endpoint
concentrations of solutions B and C are determined using
the equation in that test.
Table 1. Preparation of Solutions for the
Inhibition/Enhancement Test for Gel-Clot Techniques

Endotoxin
Initial
Concentration/S
Diluti Endotoxin Numbe
olution to which
on
r of
Soluti Endotoxin is Dilue Fact Concentr Replica
on
Added
nt
or
ation
tes
Aa

none/sample
solution

Bb

2 /sample
solution

Cc

2 /water for
BET

sampl
e
soluti
on

1
2
4
8

2
1
0.5
0.25

4
4
4
4

2
1
0.5
0.25

2
2
2
2

LAL
Reag
ent
Water

1
2
4
8

Endotoxin
Initial
Concentration/S
Diluti Endotoxin Numbe
olution to which
on
r of
Soluti Endotoxin is Dilue Fact Concentr Replica
on
Added
nt
or
ation
tes
Dd

none/LAL
Reagent Water

Solution A: a sample solution of the preparation under

test that is free of detectable endotoxins.
b

Solution B: test for interference.

Solution C: control for labeled LAL Reagent sensitivity.

Solution D: negative control of LAL Reagent Water.

This test must be repeated when any condition that is

likely to influence the test results changes. The test is not
valid unless Solutions A and D show no reaction and the
result of Solution C confirms the labeled sensitivity.
If the sensitivity of the lysate determined in the presence
of the sample solution under test of Solution B is not less
than 0.5 and not greater than 2 , the sample solution
does not contain factors which interfere under the
experimental conditions used. Otherwise, the sample
solution to be examined interferes with the test.
If the sample under test does not comply with the test at a
dilution less than the MVD, repeat the test using a greater
dilution, not exceeding the MVD. The use of a more
sensitive lysate permits a greater dilution of the sample to
be examined and this may contribute to the elimination of
interference.
Interference may be overcome by suitable treatment,
such as filtration, neutralization, dialysis, or heating. To
establish that the chosen treatment effectively eliminates
interference without loss of endotoxins, perform the assay
described below using the preparation to be examined to
which USP Endotoxin RS has been added and which has
been subjected to the selected treatment.
Gel-Clot Limit Test
This test is used when a monograph contains a
requirement for endotoxin limits.
Procedure Prepare Solutions A, B, C, and D as shown
in Table 2, and perform the test on these solutions
following the procedure in the Test for Confirmation of

Labeled LAL Reagent Sensitivity under Preparatory

Testing for the Gel-Clot Techniques.
Table 2. Preparation of Solutions for the Gel-Clot Limit
Test

Solution*

Endotoxin
Concentration/Solution to
which Endotoxin is Added

Table 3. Preparation of Solutions for the Gel-Clot Assay

Number of
Replicates

none/diluted sample solution

2 /diluted sample solution

2 /LAL Reagent Water

none/LAL Reagent Water

Procedure Prepare Solutions A, B, C, and D as shown

in Table 3, and test these solutions by following the
procedure in the Test for Confirmation of Labeled LAL
Reagent Sensitivity under Preparatory Testing for the
Gel-Clot Techniques.

Endotoxin
Concentrati
on/
Solution to
Diluti
Initial
Number
which
on Endotoxin
of
Soluti Endotoxin Diluen Facto Concentrat Replicat
on is Added
t
r
ion
es
Aa

none/sampl LAL
e solution Reage
nt
Water

Bb

2 /sample
solution

Cc

Dd

Prepare Solution A and positive product control

Solution B using a dilution not greater than the MVD and
treatments as directed in the Interfering Factors Test for
the Gel-Clot Techniques under Preparatory Testing for
the Gel-Clot Techniques. Positive control Solutions B
and C contain the standard endotoxin preparation at a
concentration corresponding to twice the labeled LAL
Reagent sensitivity. The negative control Solution D is
LAL Reagent Water.
Interpretation The test is not valid unless both
replicates of positive control Solutions B and C are
positive and those of negative control Solution D are
negative. The preparation under test complies with the
test when a negative result is found for both tubes
containing Solution A. The preparation under test does
not comply with the test when a positive result is found for
both tubes containing Solution A.
Repeat the test when a positive result is found for 1 tube
containing Solution A and a negative result for the other
one. The preparation under test complies with the test
when a negative result is found for both tubes containing
Solution A in the repeat result. If the test is positive for the
preparation under test at a dilution less than the MVD, the
test may be repeated at a dilution not greater than the
MVD.
Gel-Clot Assay
This assay quantifies bacterial endotoxins in sample
solutions by titration to an endpoint.

1
2
4
8

2
2
2
2

2 /LAL
Reagent
Water

LAL
Reage
nt
Water

1
2
4
8

2
1
0.5
0.25

2
2
2
2

none/LAL
Reagent
Water

Solution A: a sample solution under test at the dilution,

not to exceed the MVD, with which the Interfering
Factors Test for the Gel-Clot Techniques was completed.
Subsequent dilution of the sample solution must not
exceed the MVD. Use LAL Reagent Water to make
dilution series of four tubes containing the sample
solution under test at concentrations of 1, , , and 1/8
relative to the dilution with which the Interfering Factors
Test for the Gel-Clot Techniques was completed. Other
dilutions may be used as appropriate.
b

Solution B: Solution A containing standard endotoxin

at a concentration of 2 (positive product control).
c

Solution C: two series of 4 tubes of LAL Reagent

Water containing the standard endotoxin at a

Endotoxin
Concentrati
on/
Solution to
Diluti
Initial
Number
which
on Endotoxin
of
Soluti Endotoxin Diluen Facto Concentrat Replicat
on is Added
t
r
ion
es
concentration of 2 , , 0.5 , and 0.25 , respectively.
d

Solution D: LAL Reagent Water (negative control).

Calculation and Interpretation The test is not valid

unless the following conditions are met: (1) both
replicates of negative control Solution D are negative; (2)
both replicates of positive product control Solution B are
positive; and (3) the geometric mean endpoint
concentration of Solution C is in the range of 0.5 to 2 .
To determine the endotoxin concentration of Solution A,
calculate the endpoint concentration for each replicate
series of dilutions by multiplying each endpoint dilution
factor by . The endotoxin concentration in the sample is
the geometric mean endpoint concentration of the
replicates (see the formula given in the Test for
Confirmation of Labeled LAL Reagent Sensitivity under
Preparatory Testing for the Gel-Clot Techniques). If the
test is conducted with a diluted sample solution, calculate
the concentration of endotoxin in the original sample
solution by multiplying by the dilution factor. If none of the
dilutions of the sample solution is positive in a valid
assay, report the endotoxin concentration as less than
(if the diluted sample was tested, less than times the
lowest dilution factor of the sample.) If all dilutions are
positive, the endotoxin concentration is reported as equal
to or greater than the greatest dilution factor multiplied by
(e.g., initial dilution factor times 8 times in Table 3).
The article meets the requirements of the test if the
concentration of endotoxin is less than that specified in
the individual monograph.
PHOTOMETRIC TECHNIQUES
The turbidimetric method measures increases in turbidity.
Depending on the test principle used, this technique is
classified as either endpoint-turbidimetric or kineticturbidimetric. The endpoint-turbidimetric technique is
based on the quantitative relationship between the
concentration of endotoxins and the turbidity (absorbance

or transmission) of the reaction mixture at the end of an

incubation period. The kinetic-turbidimetric technique is a
method to measure either the onset time needed to reach
a predetermined absorbance of the reaction mixture or
the rate of turbidity development.
The chromogenic method measures the chromophore
released from a suitable chromogenic peptide by the
reaction of endotoxins with the LAL Reagent. Depending
on the test principle employed, this technique is classified
as either endpoint-chromogenic or kinetic-chromogenic.
The endpoint-chromogenic technique is based on the
quantitative relationship between the concentration of
endotoxins and the release of chromophore at the end of
an incubation period. The kinetic-chromogenic technique
is a method to measure either the onset time needed to
reach a predetermined absorbance of the reaction
mixture or the rate of color development.
All photometric tests are carried out at the incubation
temperature recommended by the LAL Reagent
manufacturer, which is usually 37 1 .
Preparatory Testing for the Photometric Techniques
To assure the precision or validity of the turbidimetric and
chromogenic techniques, preparatory tests are conducted
to verify that the criteria for the standard curve are valid
and that the sample solution does not inhibit or enhance
the reaction. Revalidation for the test method is required
when conditions that are likely to influence the test result
change.
Verification of Criteria for the Standard Curve Using the
Standard Endotoxin Solution, prepare at least three
endotoxin concentrations to generate the standard curve.
Perform the test using at least three replicates of each
standard endotoxin concentration according to the
manufacturer's instructions for the LAL Reagent (with
regard to volume ratios, incubation time, temperature, pH,
etc.). If the desired range in the kinetic methods is greater
than two logs, additional standards should be included to
bracket each log increase within the range of the
standard curve. The absolute value of the correlation
coefficient, |r|, must be greater than or equal to 0.980 for
the range of endotoxin concentrations indicated by the
manufacturer of the LAL Reagent.
Interfering Factors Test for the Photometric Techniques
Select an endotoxin concentration at or near the middle
of the endotoxin standard curve. Prepare Solutions A, B,
C, and D as shown in Table 4. Perform the test on
Solutions A, B, C, and D at least in duplicate following the

instructions for the LAL Reagent used (with regard to

volume of sample and LAL Reagent, volume ratio of
sample to LAL Reagent, incubation time, etc.).
Table 4. Preparation of Solutions for the
Inhibition/Enhancement Test for Photometric Techniques

Solution

Endotoxin
Concentration

Solution to
which
Endotoxin is Number of
Added
Replicates

Aa

none

sample
solution

not less
than 2

Bb

middle
concentration of
the standard curve

sample
solution

not less
than 2

concentration after subtraction of any endotoxin detected

in the solution without added endotoxin.
When the endotoxin recovery is out of the specified
ranges, the interfering factors must be removed as
described in the Interfering Factors Test for the Gel-Clot
Techniques under Preparatory Testing for the Gel-Clot
Techniques. Repeating the Interfering Factors Test for the
Gel-Clot Techniques validates the treatment.
Procedure for the Photometric Techniques
Follow the procedure described in the Interfering Factors
Test for the Photometric Techniques under Preparatory
Testing for the Photometric Techniques.
Calculation for the Photometric Techniques

Solution A: the sample solution may be diluted not to

exceed MVD.

Calculate the endotoxin concentration of each of the

replicates of test Solution A using the standard curve
generated by positive control series C. The test is not
valid unless the following conditions are met: (1) the
results of control series C comply with the requirements
for validation defined under Verification of Criteria for the
Standard Curve under Preparatory Testing for the
Photometric Techniques; (2) the endotoxin recovery,
calculated from the concentration found in Solution B
after subtracting the endotoxin concentration found in
Solution A is within 50 to 200%; and (3) the result of
negative control series D does not exceed the limit of the
blank value required in the description of the LAL
Reagent used.

Interpretation of Results from the Photometric Techniques

Cc

at least 3
concentrations
(lowest
concentration is
designated )

LAL Reagent each not

Water
less than 2

Dd

none

LAL Reagent
Water

not less
than 2

Solution B: the preparation under test at the same

dilution as Solution A, containing added endotoxin at a
concentration equal to or near the middle of the standard
curve.
c

Solution C: the standard endotoxin at the

concentrations used in the validation of the method
described in Verification of Criteria for the Standard
Curve under Preparatory Testing for the Photometric
Techniques (positive control series).
d

Solution D: LAL Reagent Water (negative control).

Calculate the mean recovery of the added endotoxin by

subtracting the mean endotoxin concentration in the
solution (if any) from that containing the added endotoxin.
In order to be considered free of interfering factors under
the conditions of the test, the measured concentration of
the endotoxin added to the sample solution must be
within 50% to 200% of the known added endotoxin

In photometric assays, the preparation under test

complies with the test if the mean endotoxin
concentration of the replicates of Solution A, after
correction for dilution and concentration, is less than the
endotoxin limit for the product.

LAL Reagent reacts with some -glucans in addition

to endotoxins. Some preparations that are treated will not
react with -glucans and must be used for samples that
contain glucans.
2

For a validity test of the procedure for inactivating

endotoxins, see Dry-Heat Sterilization under Sterilization
and Sterility Assurance of Compendial Articles 1211 .
Use an LAL Reagent having a sensitivity of not less than
0.15 Endotoxin Unit per mL.

Sterile Water for Injection or other water that shows

no reaction with the specific LAL Reagent with which it is
to be used, at the limit of sensitivity of such reagent.
4

K is 5 USP-EU/kg for any route of administration

other than intrathecal (for which K is 0.2 USP-EU/kg body
weight). For radiopharmaceutical products not
administered intrathecally the endotoxin limit is calculated
as 175/V, where V is the maximum recommended dose
in mL. For intrathecally administered
radiopharmaceuticals, the endotoxin limit is obtained by
the formula 14/V. For formulations (usually anticancer
products) administered on a per square meter of body

surface, the formula is K/M, where K = 5 EU/kg and M is

the (maximum dose/m2/hour 1.80 m2)/70 Kg.
Auxiliary Information Staff Liaison : Radhakrishna S
Tirumalai, Scientist
Expert Committee : (MSA05) Microbiology and Sterility
Assurance
USP29NF24 Page 2521
Phone Number : 1-301-816-8339