Lyophilized LAL should be stored at 2-8ºC; avoid exposure to temperatures

above 25ºC. The tube should be discarded if there is any yellow discoloration.

REAGENTS AND MATERIALS NOT PROVIDED

LIMULUS AMEBOCYTE LYSATE LAL Reagent Water (non LAL-reactive) must be used to prepare samples and
positive controls. See Product No. W110.
ENDOSAFE® [Link] Control Standard Endotoxin (CSE) is available from Charles River
U.S. License No. 1197 Endosafe to confirm LAL reagent sensitivity, validate product test methods,
and prepare inhibition controls (positive water and positive product controls).
Refer to the Certificate of Analysis for each CSE lot for potency, rehydration,
SINGLE-TEST Vial For Endotoxin (Pyrogen) Detection and storage information. CSE must be ordered separately for this product.
High and low potency CSE are available from Endosafe.
INTENDED USE: Limulus amebocyte lysate (LAL),
A water bath or heating block is required to incubate the assay mixture at a
derived from Limulus polyphemus amebocytes,is intended
temperature of 37º C, plus or minus 1º C. Sterile, endotoxin-free accessories
for use in the qualitative detection of gram-negative are needed which include: 16 x 125 mm or larger reusable borosilicate tubes
bacterial endotoxins by the gel-clot method. or equivalent, a calibrated mechanical pipetor with sterile, disposable plastic
tips for accurate delivery of volumes less than 1 mL, and pipets for larger
volumes. Test tube racks are needed for holding reaction tubes and standard
endotoxin dilution tubes. Timers are useful in measuring incubation times and
endotoxin mixing periods.

REAGENT PREPARATION: Caution: Single-test LAL must be incubated

SUMMARY AND GENERAL INFORMATION: The LAL test is the most immediately after rehydration with test sample or control. The LAL is
reconstituted by addition, directly into the test vial, of 0.2 mL of control

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sensitive and specific means to detect and measure endotoxin, a fever-producing
byproduct of gram-negative bacteria commonly known as pyrogen. The basis solution or sample to be tested.
of the test is that endotoxin produces an opacity and gelation in LAL that is
readily recognized.5 The simplicity and economy of the LAL Test encourages PREPARATION OF CONTROL STANDARD ENDOTOXIN (CSE)

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the testing of in-process solutions and raw materials as well as end-product Reconstitution: A CSE of E. coli is available from Charles River Endosafe
drugs, devices and biologics.6 The USP Bacterial Endotoxins Test and U.S. which is suitable for confirmation of LAL labeled sensitivity and for
Food and Drug Administration Guideline for LAL testing provide standard preparation of positive controls. The CSE has a predetermined amount of
methods for validating the LAL Test as a replacement for the rabbit pyrogen endotoxin, as described in the Certificate of Analysis (COA), which was
test.10,11 standardized with U.S. Reference Endotoxin. Note that the COA is specific to
a lysate lot and CSE lot. The USP Reference Standard Endotoxin may be pur-

O
The gel-clot LAL test method is a simple, reproducible, test that is conducted chased from the U.S. Pharmacopoeial Convention, Inc., Rockville, MD 20852.
by mixing ENDOSAFE® LAL reagent and test specimen and promptly The lyophilized endotoxin (CSE) must be prepared according to the package
incubating the mixture undisturbed for 60 minutes at 37ºC. A positive insert and the COA. Rehydrate the CSE with LAL Reagent Water and vortex
response on the gel clot test indicates there is an amount of endotoxin in the sam- vigorously for 5 minutes before further dilution. Dilution to 1
ple which equals or exceeds the reagent’s labeled sensitivity, represented by the EU/mL should be made, then two-fold dilutions to bracket the labeled LAL
symbol lambda, λ. Reagent sensitivity.
n
Storage: Rehydrated endotoxin may be stored for 28 days at 2 to 8º C. Diluted
BIOLOGICAL PRINCIPLES: The development of a viable alternative to the
endotoxin solutions should be made daily unless longer intervals have been
rabbit pyrogen test began with the innovative work of John Hopkins Univ.
validated.
investigators. Frederick Bang observed that bacteria caused intravascular
coagulation in the American horseshoe crab, Limulus polyphemus. In
io
SPECIMEN COLLECTION AND PREPARATION
collaboration, Levin and Bang5 found that the agent responsible for the clotting
Specimen for testing with Endosafe® LAL must be collected and prepared using
phenomena resided in the crab’s amebocytes, or circulating blood cells, and
depyrogenated materials and endotoxin-free reagents. If the positive product
that pyrogen (bacterial endotoxin) produced a gelation reaction of amebocyte
control fails and a pH related problem is suspected, the pH of the test specimen
lysate by an enzymatic process. Serine protease zymogens found in amebocyte
and LAL mixture should be measured to assure a pH within the range of pH
lysate are activated by endotoxin in the presence of divalent cations to initiate
at

6.0 to 8.0. If pH adjustment is necessary, use endotoxin-free HCl or NaOH at a

an enzymatic coagulation cascade that alters an abundant protein called
suitable concentration (generally 0.1N or less), or a Tris buffer from Charles
coagulogen to produce a proteinaceous gel.7
River Endosafe. Do not arbitrarily adjust the pH of unbuffered solutions. If
the specimen contains interfering substances, dilute or modify the specimen to
The need for a suitable pyrogen test for radiopharmaceuticals led Cooper,
an extent that eliminates interference, as discussed in the PRODUCT
Levin and Wagner to extend this new approach to drugs. A comparative study
INHIBITION Section.
in 1970 demonstrated that the LAL test was more sensitive than the rabbit test
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and that LAL reactivity (gelation and increased opacity) correlated with
TEST PROCEDURE: The single-test vials containing LAL serve as the test
endotoxin concentration.3 Improvements in LAL reagents, the advent of
container. Before use, collect the vial contents by gently tapping the bottom
standard methods and automated systems, and a better understanding of LAL
of the vial on a hard surface. Use aseptic technique when removing the rubber
reactivity make the LAL reagent readily adaptable to testing a variety of
stopper. Test as follows:
biologics, parenteral products and medical devices.2,4,9
Aseptically add 0.2 mL of each test specimen to assay tubes. Mix the con-
The LAL reaction requires a neutral pH and is time and concentration depend-
tents gently until the contents are dissolved. Immediately place the reaction
ent. The test is generally limited to aqueous solutions or extracts of test spec-
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tubes in a 37ºC water or dry bath for 60 minutes (plus or minus 2 minutes).
imen. Most LAL test interferences are overcome by simple dilution.8
Timing of the reaction of ENDOSAFE® LAL with endotoxin is critical. If
large numbers of samples are to be tested in parallel, the reactions should be
USFDA GUIDELINE FOR END PRODUCT TESTING
started at 2-4 minute intervals so as to permit reading of each test within the
A guideline was released by the U.S. Food and Drug Administration in 1987 to
above time limit.
inform manufacturers of human drugs and biologicals, animal drugs, and
medical devices of procedures the Agency considers necessary to validate the
In

Since the reaction of ENDOSAFE® LAL is temperature sensitive, the incubator

use of LAL as an end-product endotoxin test.10 Those who adhere to the
must be monitored carefully. Also, the gel-forming reaction is delicate and
guideline are considered in compliance with relevant cGMP provisions for
may be irreversibly altered if the tubes are disturbed during the incubation
drugs and devices and other applicable requirements. The general endotoxin
period.
limit for parenteral drugs is 5 Endotoxin Units (EU) per Kg dose, except for
a 0.2 EU/Kg limit for intrathecal drugs. Medical device eluates must not
ENDOTOXIN CONTROL SERIES (Positive Water Controls)
exceed 0.5 EU/mL; a 0.06 EU/mL limit applies to devices that contact
An endotoxin standard series does not have to be run with each set of tests if
cerebrospinal fluid.10
consistency of standard endpoints has been demonstrated. It should be run at
least once a day with the first set of tests and repeated if there is any change
GENERAL PRECAUTIONS: ENDOSAFE® LAL is intended for in vitro
in LAL lot or test conditions.
diagnostic purposes only. It is not to be used for detection of endotoxemia.
Avoid direct contact with LAL because its toxicity is not known.
A fresh CSE control dilution series should be prepared from a stock solution
in a two-fold dilution series that brackets the labeled sensitivity (λ) of
Correct application of this test requires strict adherence to all items in the
recommended procedures. Positive controls should be included in LAL ENDOSAFE® LAL reagent. A 4-point series is usually made with two
protocols to detect inhibitory conditions. All materials coming in contact with endotoxin dilutions above and below. Add 0.2 mL of each concentration of
specimen or test material must be endotoxin-free. Glassware must be endotoxin directly to the assay tube. Mix and incubate as described above.
depyrogenated by validated conditions, such as three hours exposure at 200º TEST CONTROLS: Prepare the Negative Control by adding 0.2 mL of the
C. It is our experience that plasticware labeled as sterile and disposable is LAL Reagent Water to the assay tube. In the absence of an endotoxin series,
endotoxin-free. add 0.2 mL of a 2 lambda concentration of the endotoxin standard. For a
Positive Product Control, add 0.2 mL of a mixture containing a 2 lambda
REAGENTS PROVIDED concentration of endotoxin in the test specimen, which may be modified or
Lyophilized LAL (ENDOSAFE®) is presented in single-test glass vials. The diluted consistent with validated conditions. This control assures the absence
reagent contains buffered lysate and is stabilized by monovalent and divalent of interference. See section on PRODUCT INHIBITION.
cations, and is sealed under approximately 1/2 atmosphere of dry nitrogen. Do
not rehydrate until immediately prior to use.
INTERPRETATION OF RESULTS: Each tube in the gel-clot method is particularly resistant to interference because of its high buffer capacity and
interpreted as either positive or negative. A positive result is defined as the balanced divalent and monovalent cation formulation.
formation of a firm gel capable of maintaining its integrity when the test tube
is inverted 180º. A negative test is characterized by the absence of gel or by Maximum Valid Dilution: The U.S. Food and Drug Administration has
the formation of a viscous mass which does not hold when the tube is inverted. established endotoxin limits of 5 EU/Kg for intravenous drugs and 0.2 EU/Kg
Test results are only valid when the positive water and specimen controls are for intrathecal drugs.10 The U.S. Pharmacopeia has adopted specific limits for
positive at the 2 lambda endotoxin concentration, and the negative controls are compendial items such as 175 EU per dose of radiopharmaceutical.11 These
without gelation. limits may be used to determine the extent of dilution that may be applied to
overcome an interference problem without exceeding the limit endotoxin
EXPECTED VALUES: ENDOSAFE® LAL Reagent is standardized against the concentration. The Maximum Valid Dilution (MVD) may be calculated by
U.S. Reference Endotoxin, so that the sensitivity is expressed in Endotoxin formulae presented in the previously mentioned documents.
Units per milliliter (EU/mL). Confirmation of label claim is an assay of the
LAL by a standardized control endotoxin which yields an endpoint that is equal For drug products that have a published limit, the MVD may be calculated by
to or within a two-fold dilution of the labeled sensitivity. The results of an the following formula:
endotoxin assay of a LAL Reagent labeled with a sensitivity (λ) of 0.125
EU/mL is presented in Table I. A 4-point endotoxin dilution series was MVD = Endotoxin Limit x Potency of Product
prepared to bracket the labeled sensitivity. Labeled Sensitivity, λ
TABLE I: CONFIRMATION OF LABEL CLAIM ASSAY
For example, the compendial limit for cyclophosphamide is 0.17 EU/mg. If a
Endotoxin Dilution (EU/mL) LAL Reagent with λ = 0.125 is used to test this product where the potency is
Replicate 0.25 0.125 0.06 0.03 Endpoint
1 + + + - 0.06
20 mg/mL, the MVD equals 1:27.
2 + + - - 0.125
3 + + - - 0.125 MVD = 0.17 EU/mg x 20 mg/mL = 27.2
4 + + + - 0.06 0.125 EU/mL

y
Under these conditions, cyclophosphamide may be diluted up to 1:27 in order
The LAL sensitivity is calculated by determining the geometric mean of the to resolve an inhibition that might be present.
endpoint. Each endpoint of the quadruplicate assay is converted to log10. The
LIMITATIONS: Samples may be tested by LAL methods provided that no
individual log10 values are averaged and the LAL sensitivity is taken as the

nl
inhibition or enhancement conditions are present that cannot be eliminated by
antilog of this average log value (see Table ΙΙ). an acceptable dilution (refer to MVD calculation) or sample-pretreatment, such
TABLE II: CALCULATION OF GEOMETRIC MEAN ENDPOINT as buffering. If the LAL method cannot be validated at a concentration within
Endpoint (EU/mL) Log10 Endpoint the maximum valid dilution, the LAL test cannot be substituted for the USP
0.06 -1.222 Pyrogen Test.10

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0.125 -0.903 The error of the gel-clot method is plus or minus one two-fold dilution of the
0.125 -0.903 endpoint of the assay.
0.06 -1.222
Mean= -1.0625
Antilog10 Mean= 0.0865
BIBLIOGRAPHY
1. Bang, F.B. “A Bacterial Disease of Limulus Polyphemus,” Bull. Johns Hopkins
INITIAL QUALITY CONTROL PROCEDURE FOR A TESTING Hosp., 98, p.325 (1956).
LABORATORY: The variability of a test laboratory and its analysts should
n
be assessed before any official tests are done. Each analyst, using a single lot 2. Cooper, J.F. and Harbert, J.C. “Endotoxin as a Cause of Aseptic Meningitis after
of LAL and a single lot of endotoxin (CSE or RSE), should correctly and Radionuclide Cisternography.” J. Nucl. Med., 16, p.809 (1976).
satisfactorily complete the test for confirmation of labeled LAL sensitivity.
Acceptable variation is one half (0.5 λ) to two times (2 λ) labeled sensitivity (λ). 3. Cooper, J.F., Levin. J., and Wagner, H.N.“Quantitative Comparison of In Vitro and
io
In Vivo Methods for the Detection of Endotoxin,” J. Lab. Clin. Med., 78, p.138 (1971).
TEST FOR CONFIRMATION OF LABELED LAL REAGENT
SENSITIVITY: The labeled sensitivity must be confirmed before a new LAL 4. Hochstein, H.D. The LAL test versus the rabbit pyrogen test for endotoxin detection:
lot is introduced into a test laboratory. A single lot of LAL should be assayed Update ’87. Pharm. Technol., 11(6), p.124 (1987).
by a single lot of endotoxin (CSE or RSE) by testing in quadruplicate vials (see
Table I). The geometric mean of the endpoints must be within the limits of 5. Levin, J. and Bang, F.B., “Clottable Protein in Limulus: Its Localization and Kinetics
at

labeled claim, as defined and illustrated above. of Its Coagulation by Endotoxin.” Thromb. Diath. Haemorrh., 19, p.186 (1968).

DETERMINATION OF ENDOTOXIN IN AN UNKNOWN: 6. McCullough, K.Z. Process control: In-process & raw material testing using LAL.
To determine the endotoxin concentration in an unknown, test serial two-fold Pharm. Technol., 12(5) p.40 (1988).
dilutions of the specimen until an endpoint is reached. The endotoxin
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concentration (E) in a sample is calculated by multiplying the LAL labeled 7. Tai, J.Y. and Liu, T.Y. Studies on Limulus lysate. Isolation of pro-clotting enzyme.
sensitivity by the reciprocal of the dilution representing the endpoint. For J. Biol. Chem., 252, p.2178 (1977).
example, a product aliquot was diluted by preparing a series of two-fold
dilutions with LAL Reagent Water. A test of each product dilution yielded an 8. Cooper, J.F. “Resolving LAL Test Interferences.” J. Parent. Sci. & Tech., 44:1, p.13
endpoint at the 1:8 dilution when tested with LAL Reagent having a labeled (1990).
sensitivity (λ) equal to 0.25 EU/mL. The endotoxin titer was determined to
contain at least 2 EU/mL by the following calculation: 9. Weary, M. Pyrogen testing with the Limulus amebocyte lysate test. Pharmacy Int.,
(E) = ( λ )(8/1) = (0.25 EU/ml)(8) = 2 EU/ml 7:99.
fo

PRODUCT INHIBITION: Before routine LAL testing is started, the potential 10. Guideline on Validation of the Limulus Amebocyte Lysate Test as an End-
for product inhibition must be excluded. Inhibition is usually concentration Product Endotoxin Test for Human and Animal Parenteral Drugs, Biological
dependent, and is easily overcome by dilution with LAL Reagent Water. Products, and Medical Devices. U.S. Dept. of Health & Human Services, FDA,
Common sources of inhibition include conditions that 1) interfere with the December 1987.
enzyme-mediated gelation reaction, and 2) alter the dispersion of the endotoxin
11. Bacterial endotoxins test. In The U.S. Pharmacopeia, 23rd revision, p.1696-1697.
In

control. Inhibition exists if the endpoint of an assay of a two-fold endotoxin

dilution series made with the specimen (Positive Product Controls) differs more Mack Publishing Co., Easton, PA 1995.
than one two-fold dilution from the endpoint of a similar endotoxin series in
water (Positive Water Control). Product inhibition may be recognized as SIGNIFICANT TEXTS ON LAL TESTING
follows: Bacterial Endotoxins: Structure, Biomedical Significance, and Detection With the Limulus
Amebocyte Lysate Test. Editors: J.W. ten Cate, H.R. Buller, A. Sturk and J. Levin.
Labeled LAL Sensitivity ( λ ) = 0.125 EU/mL Progress in Clinical and Biological Research, Vol. 189, Alan R. Liss, Inc., NY, 1985.
Endpoint Positive Water Controls = 0.125 EU/mL
Endpoint Positive Product A Controls = 0.20 EU/mL Pearson, F.C. Pyrogens: Endotoxins, LAL Testing, and Depyrogenation. Marcel Dekker,
Endpoint Positive Product B Controls = 0.50 EU/mL New York and Basal, 1985.

Product A is considered within limits whereas Product B exhibits inhibition. Endotoxins and their Detection with the Limulus Amebocyte Lysate Test. Editors: S.
The easiest method to determine the non-inhibitory product concentration is to Watson, J. Levin, T. Novitsky). Progress in Clin. Biol. Res., 93, Alan R. Liss, New York,
prepare a series of increasing dilutions of the product containing a 2 lambda 1982.
endotoxin concentration.8,10 Assay this series as well as a series of the product
diluted with water. The following results are consistent with a product that is
non-inhibitory at a 1:20 dilution or greater, and is endotoxin-free.
Manufactured By: PRODUCT CODES
Specimen Dilution 1:4 1:10 1:20 1:40 CHARLES RIVER ENDOSAFE - U.S. License No. 1197
Product and 2 λ Endotoxin - -+ ++ ++ Div. of Charles River Laboratories, Inc. R13003
Product and LAL Reagent Water - - - - 1023 Wappoo Road, 43-B R13006
Charleston, SC 29407, USA R13012
Raw materials may be acidic or basic and require pH adjustment to neutrality R13025
as well as dilution to resolve product inhibition. Endosafe LAL reagent is PIR13000