Le DEPARTMENT OF HEALTH & HUMAN SERVICES Public Health Service
2 ——__. —. ee nn
a. July 15, 1993

Food and Drug Administration
Interim Guidance for Human and Veterinary Drug Products and Beeeilg MQ 2857

KINETIC LAL TECHNIQUES

according to the lysate manufacturers recommended Procedures, i.e., sample/lysate
Tatia, incubation temperature and times, measurement wavelength, etc.
Instrumentation other than the one recommended by lysate manufacturer can be
used. The performance characteristics (slope, y-intercept and correlation
coefficient), for the lysate lot, sent by the manufacturer will not be valid.
New performance characteristic have to be established for each lot by performing
the procedures outlined in Appendix A.

BITIO} IC iN’ STING

In inhibition/enhancement testing of a Product by kinetic techniques, test a drug
concentration containing a quantity of the RSE or CSE between 0.1 and 0.5 EU/aL
or 1.0 and 5.0 EU/mL depending on its Pass/ Fail cutofe (PFC) in duplicate
according to the lysate manufacturer ‘e methodology. The 4 lambda spike
Procedure, in the current guideline, is still valid and can be used in the
kinetic techniques. This procedure should be used with caution if lambda is leas
than 0.01 EU/mL.

between 0.1 and 0.S EU/mL, otherwise the endotoxin spike should be between 1.0

of the range. The standard curve mast meet the criteria outlined in Appendix A.
The calculated mean amount of endotoxin when referenced to the standard curve,

inhibition or enhancement, the drug product can be diluted, not exceeding the
MVD, and test repeated.

An alternate Procedure may be used, in which the RSE/CSE atandard is prepared in
drug product or product dilution instead of water. The drug Product (at the
concentration used to prepare the standard curve), cannot have an endotoxin
concentration greater than the lowest concentration used to generate the product
standard curve, when referenced against a standard curve Prepared in water. The
Product standard curve must Meet the test for linearity, i.e. 5 equal to or
greater than the abaolute value of 0.980, and Slope of the regression line must
be less than ~0.1 and greater than -1.0. r¢ the standard curve does not meet
these Criteria, the drug product Cannot be tested by the alternate procedure.

ROUTINE TESTING
SQUTINE TESTING

in the range of the standard curve go that there is at least one Standard per log
increment of the range. The standard curve must meet the criteria outlined in
Appendix A. For the kinetic techniques, it ia not necessary to run a standard


Immune Serum Globulin 5.50 mL

Indigotindisulfonate Sodium Injection 1.00 mL
Indium Inl11 Oxyquinoline 7.00 mL
*Indium Pentetate In111 Injection 0.50 mL
Indium Chlorides In113m Injection 2.00 mL
Indocyanine Green 0.70 mg
Indomethacin Sodium 0.20 mg
Insulin 2.00 units
Insulin Human oe
Interferon Alfa-n1 77142.00 units
Interferon Alfa-n3 3571.00 units
Interferon Alfa - 2a 428571.00 units
Interferon Alfa - 2b 514285.00 units
Inulin 50.00 mg
Invert Sugar 2.38 mL
Jodamide meglumine - 24% 4.30 mL
Iodide Sodium 1123 Solution 7.00 mL
Iodinated 1125 Albumin Injection 7.00 mL
Iodide Sodium 1125 Solution 7.00 mL
Todinated 1131 Albumin Injection 7.00 mL
Todinated 1131 Albumin Aggregated Injection 7.00 mL
Iodohippurate Sodium 1131 Injection 7.00 mL
Rose Bengal Sodium [131 Injection 7.00 mL
Iodide Sodium [131 Solution 7.00 mL
Iodipamide Meglumine Injection - 52% 0.60 mL

- 10.5% 1.40 mL
Iodipamide Meglumine -

Diatrizoate meglumine 0.14 mL
*Iohexol 43.70 mg I
*Iopamidol 40.00 mg I
*Iophendylate Injection 0.22 mL
Iothalamate Meglumine Injection 80%- 1.40 mL

60%- 2.00 mL
43%- 5.70 mL
30%- 4.30 mL

17.2% -5.70 mL
Iothalamate Meglumine -

Iothalamate Sodium 52%-26% 1.50 mL
Tothalamate Sodium 66.8% - 1.50 mL
54.3% - 0.90 mL

Toxaglate Meglumine 3.00 mL

0.91
5.00 +

25.00 +

28.00 +

87.50 +
7.10 +

25.00
2.50
0.80 +
0.65/10,000 #
0.14/100 #
0.10/10,000 #
0.10/10,000 #
0.10 +
2.10
1.20

25.00 +

25.00 +

25.00 +

25.00 +

25.00 +

25.00 +

25.00 +

25.00 +
8.33
3.60 +

35.71
0.11
0.6 +
0.90 +
3.57
2.50
0.90 +
1.16
0.90

3.35 +
3.35 +
5.56
1.67


Diatrizoate Meglumine Injection 60%
30%
Diatrizoate Meglumine
and Diatrizoate Sodium 66% - 10%
60% - 30%
52% - 8%
50% - 25%
34.3% - 35%
28.5% - 29.1%
Diatrizoate Meglumine
and Iodipamide Meglumine
Diatrizoate Sodium 50%
25%
20%
Diazepam Injection
Diazoxide Injection
*Dibucaine
Dibucaine HCI and Dextrose
Dicloxacillin Sodium
Dicyclomine HCI Injection
Diethylstilbestrol Injection
Diethylstilbestrol Diphosphate
Digitoxin Injection
Digoxin Injection
Digoxin Immune Fab
Dihydroergotamine Mesylate
Dihydroergotamine Mesylate, Heparin
Sodium & Lidocaine HCI

Dihydrostreptomycin Sulfate
Dihydrotachysterol

Diluent for Meningococcal Vaccine
Dimenhydrinate Injection
Dimethyl! Sulfoxide Irrigation
Dimercaprol

Dinoprost Tromethamine
Diphenhydramine HCI Injection
Diphenidol

Diphtheria Antitoxin, Pur.Conc. (equine)
Dipyridamole

Dobutamine HCI

1.00 mL
4.40 mL

2.30 mL
1.40 mL
2.80 mL
2.80 mL
2.80 mL
2.80 mL

0.11 mL
1.00 mL
4.00 mL
0.90 mL
0.43 mg
10.00 mg
0.14 mg
0.07 mg
0.29 mg
0.29 mg
7.14 mg
7.14 mg
0.045 mg
0.025 mg
5.00 mg
0.014 mg

1667.00 units
(Heparin)
10.00 mg
0.03 mg
5.00 mL
1.25 mg
10.00 mL
5.00 mg
0.57 mg
1.50 mg
0.30 mg
3.00 mL
0.14 mg
0.90 mg

40

5.00
1.10

2.417
3.57
1.80
1.80
1.80
1.80

45.45
5.00
1.25
5.56

11.60 +
0.50 +

35.70 +

71.43

16.70 +

17.20 +
0.70 +
0.70 +

111.00 +
200.00 +
1.00

357.00

0.003 +

0.50 +
166.67
1.00
4.00
0.50 +
1.00
8.77
3.40 +
16.67
1.67
37.70
5.56


Plasma Protein Fraction (5%)
Plicamycin for Injection
Polyestradiol Phosphate
Polymyxin B Sulfate
*Polymyxin B Sulfate

Posterior Pituitary Injection
Potassium Acetate Injection
Potassium Chloride
Potassium Chloride,Lactated Ringers
and Dextrose Injection
Potassium Phosphate Injection
Potassium Phosphate in Dextrose
Potassium Phos., Lactated Ringers
Pralidoxine Chloride
Prednisolone Acetate Suspension
Prednisolone Acetate and
Prednisolone Sodium Phosphate
Suspension

Prednisolone Sodium Phosphate
Prednisolone Sodium Succinate
Prednisolone Tebutate Suspension
Prilocaine HCI
Prilocaine HCI and Epinephrine
Procaine HCl
Procaine HCI & Epinephrine
Procaine & Phenylephrine HCI
Procaine, Tetracaine &
Levonordefrin Injection
Propofol
Procainamide HCI Injection
Prochlorperazine Edisylate & Mesylate
Progesterone Aqueous & Suspension
Promazine HCI Injection
Promethazine HCI
Propantheline Bromide
Propiomazine HCI Injection

10.00 mL
0.05 mg
1.10 mg

1666.70 units
714.00 units

0.29 units
0.57 mEq
0.57 mEq

-.-- mL
4.43 mg
10.00 mL
10.00 mL
40.00 mg
0.16 mg

1.14 mg
Pred. Acet.
1.00 mg
0.86 mg
0.57 mg
5.70 mg
5.70 mg
8.60 mg
-.-- mg
-.-- mg

~.-- mg
12.00 mg
14.30 mg
0.28 mg
1.43 mg
2.80 mg
1.00 mg
0.43 mg
1.10 mg

Propoxycaine,Procaine HCl & Levonordefrin 6.60 mg

Propoxycaine,Procaine HCI &
Norepinephrine Bitartrate

6.60 mg

49

0.50
100.00 #
4.55
0.003
0.03/100
units
17.00 +
8.80 +
8.80 +

0.50 +
1.10 +
0.50
0.50
0.10 +
31.25

4.39

5.00 +
5.80 +
8.80 +
0.90 +
0.90 +
0.60 +
0.60 +
0.60 +

0.60 +
0.42

0.35 +
17.90 +
3.50

1.80 +
5.00 +
11.60 +
4.60 +
0.80 +

0.80 +


For the gel-clot technique, an endotoxin standard series does not
have to be run with each set of tests if consistency of standard
endpoints has been demonstrated in the test laboratory. It should
be run at least once a day with the first set of tests and repeated
if there is any change in lysate lot, endotoxin lot or test
conditions during the day. An endotoxin standard series should be

run when confirming end-product contamination. Positive product
controls ( two lamda concentration of standard endotoxin in product)
must be positive. If your test protocols state that you are using

the USP Bacterial Endotoxin Test, remember that it requires a
standard series to be run with each test. The above deviation must
be noted in your test protocol.

For the chromogenic and endpoint-turbidimetric techniques, an
endotoxin standard series does not have to be run with each set of
tests if consistency of standard curves has been demonstrated in the
test laboratory. It should be run at least once a day with the
first set of tests and repeated if there is any change in lysate
lot, endotoxin lot or test conditions during the day. However, at
least duplicates of a 4 lambda standard concentration in water and
in each product (positive product control) must be included with
each run of samples. The mean endotoxin concentration of the
standard must be within plus/minus 25% of the actual concentration
and the positive product control must meet the same criteria after
subtraction of any endogenous endotoxin. An endotoxin standard
series should be run when confirming end-product contamination. If
the alternate procedure is used, a standard in product series must
be conducted each time the product is tested.

For the kinetic-turbidimetric test, it is not necessary to run a
standard curve each day or when confirming end product contamination
if consistency of standard curves has been demonstrated in the test

laboratory.. However, at least duplicates of a 4 lambda standard
concentration in water and in each product (positive product
control) must be included with each run of samples. The mean

endotoxin concentration of the standard when calculated using an
archived standard curve (See Appendix C), must be within plus/minus
25% of the actual concentration and the positive product control
must meet the same criteria after subtraction of any endogenous
endotoxin. If the alternate procedure is used, a standard in
product series must be conducted each time the product is tested.

Before a new lot of lysate is used, the labeled sensitivity of the
lysate or the performance criteria should be confirmed by the
laboratory, using the procedures in Appendix A.

The sampling technique selected and the number of units to be tested
should be based on the manufacturing procedures and the batch size.
A minimum of three units, representing the beginning, middle, and
end, should be tested from a _ lot. These units can be run
individually or pooled. If the units are pooled and any endotoxin
is detected, repeat testing can be performed. The LAL test may be
repeated no more than twice. The first repeat consists of twice the
initial number of replicates of the sample in question to examine
the possibility that extrinsic contamination occurred in the initial

-9-


(85) BACTERIAL
ENDOTOXINS TEST

This chapter provides a test for estimating the concentration of
bacterial endotoxins that may be present in ur on the sampic of the
article(s) to which the test is applied using Limulus Amebocyte
Lysate (LAL) which has been obtained from aqueous extracts of
the circulating amebocvtes of the horseshoe crab, Limulus polv-
phemus, and which has been prepared and characterized for use
asa LAL reagent for gel-clot formation

Where the test 1s conducted as a limit test. the specimen is de-
termined to be positive or negative to the test judged against the
endotoxin concentration specified in the individual monograph
Where the test is conducted as an assay of the concentration of
endotoxin, with calculation of confidence limits of the result ob-
tained, the specimen is judged to comply with the requirements if
the result does not exceed (a) the concentration limit specified in
the individual monograph, and (b) the specified confidence limits
for the assay. In either case the determination of the reaction
end-point is made with dilutions from the material under test in
direct comparison with parallel dilutions of a reference endotoxin.
and quantities of endotoxin are expressed in defined Endotoxin

nits.

Since LAL reagents have also been formulated to be used for
turbidimetric (including kinetic assays) or colorimetric readings.

such tests may be used if shown to comply with the requirements
for alternative methods. These tests require the establishment of
a standard regression curve and the endotoxin content of the test
material is determined by interpolation from the curve. The pro-
cedures include incubation for a pre-selected time of reacting en-
dotoxin and control solutions with LAL Reagent and reading of the
spectrophotometric light absorbance at suitable wavelengths. In
the case of the turbidimetric procedure the reading is made imme-
diately at the end of the incubation period. or in the kinetic assays.
the absorbance is measured throughout the reaction period and rate
values are determined from those readings. In the colorimeiric
procedure the reaction is arrested at the end of the pre-selected time
by the addition of an appropriate amount of acetic acid solution.
prior to the readings. A possible advantage in the mathematical
treatment of results. if the test be otherwise validated and the assay
suitably designed, could be the application of tests of assay validity
and the calculation of the confidence interval and limits of potency
from the internal evidence of each assay itself (see Design and
Analysis of Biological Assays (111))

Reference Standard and Control Standard
Endotoxins

The reference standard endotoxin (RSE) is the USP Endotoxin
Reference Standard which 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,! "vortex for
not less than 20 minutes.g, and use this concentrate for making
appropriate serial dilutions. Preserve the concentrate in a refrig-
erator, for making subsequent dilutions, for not more than 14 days.
Allow it to reach room temperature, if applicable, and vortex it
vigorously for not less than 5 minutes before use.” Vortex each
dilution for not less than 1 minute before proceeding to make the
next dilution. g, Do not use stored dilutions. A control standard
endotoxin (CSE) is an endotoxin preparation other than the RSE
that has been standardized against the RSE. Ifa CSE isa prepa-
ration not already adequately characterized, its evaluation should
include characterizing parameters both for endotoxin quality and
performance (such as reaction in the rabbit), and for suitability of
the material to serve as a reference (such as uniformity and stabil-
ity). "Detailed procedures for its weighing and/or constitution
and use to assure consistency in performance should also be in-
cluded.g; Standardization of a CSE against the RSE using a LAL
Reagent for the gel-clot procedure may be effected by assaying a
minimum of 4 vials of the CSE or 4 corresponding aliquots, where
applicable, of the bulk CSE and | vial of the RSE, as directed under
Test Procedure, but using 4 replicate reaction tubes at each level

TLAL Reagent Water—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

of the dilution series for the RSE and 4 replicate reaction tubes
similarly for each vial or aliquot of the CSE. {fall of the dilutions
for the 4 vials or aliquots of the CSE cannot be accommodated with
the dilutions for the [ vial of the RSE on the same rack for incu-
bation, additional racks may be used for accommodating some of
the replicate dilutions for the CSE, but all of the racks containing
the dilutions of the RSE and the CSE are incubated as a block.
However, in such cases, the replicate dilution series from the | vial
of the RSE are accommodated together on a single rack and the
replicate dilution series from any ane of the 4 vials or aliquots of the
CSE are not divided between racks. ©The antilog of he difference
between the mean logig end-point of the RSE and the mean logio
end-point of the CSE is the standardized potency of the CSE which
then is to beg, converted to and cxpressed in Units per ag under
stated drying conditions for the CSE. or in Units per container,
whichever is appropriate. Standardize cach new lot of CSE prior
to use in the test. Calibration of a CSE in terms of the RSE must
be with the specific lot of LAL Reagent and the test procedure with
which it is to be used. Subsequent lots of LAL Reagent from the
same source and with similar characteristics need only checking of
the potency ratio. "The inclusion of one or more dilution series
made from the RSE when the CSE is used for testing will enable
observation of whether or not the relative potency shown by the
latter remains within the determined confidence limits.,~, A large
lot of a CSE may, however, be characterized by a collaborative assay
of a suitable design to provide a representative relative potency and
the within-laboratory and between-laboratory variance. "gy

A suitable CSE has a potency of not less than 2 Endotoxin Units
per ng and not more than SO Endotoxin Units per ng, where in bulk
form, under adopted uniform drying conditions, e.g.. toa particular
low moisture content and other specified conditions of use, and a
potency within a corresponding range where filled in vials of a ho-
mogencous lot.

Preparatory Testing

Use a LAL reagent of confirmed label or determined sensitivity.
In addition, where there is to be a change in lot of CSE, LAL Re-
agent or another reagent, conduct tests of a prior satisfactory lot
of CSE, LAL and/or other reagent in parallel on changeover
Treat any containers or utensils employed so as to destroy extra-
neous surface endotoxins that may be present, such as by heating
in an oven at 250° or above for sufficient time.?

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 testing untreated
specimens or appropriate dilutions thereof, concomitantly with and
without known and demonstrable added amounts of RSE or a CSE,
and comparing the results obtained. 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.

Test for confirmation of labeled LAL Reagent sensitivity —
“Confirma, the labeled sensitivity of the particular LAL reagent
with the RSE (or CSE) using not less than 4 replicate vials, under
conditions shown to achieve an acceptable variability of the test,
viz., the antilog of the geometric mean logo lysate gel-clot sensitivity
is within 0.5A to 2.0A, where ) is the labeled sensitivity in Endotoxin
Units per mL." The RSE (or CSE) concentrations selected in

confirming the LAL reagent label potency should bracket the stated
sensitivity of the LAL reagent. Confirm the labeled sensitivity of
cach new lot of LAL reagent prior to use in the test.

Inhibition or Enhancement Test—Conduct assays with standard
endotoxin, of untreated specimens in which there is no endogencous
endotoxin detectable, and of the same specimens to which endotoxin
has been added. as directed under Test Procedure, but using not
less than 4 replicate reaction tubes at each level of the dilution series
for each untreated specimen and for each specimen to which en-
dotoxin has been added. Record the end-points (E, in Units per
mL) observed in the replicates. Take the logarithms (e) of the
end-points, and compute the geometric means of the log end-points
for the RSE (or CSE), for the untreated specimens and for speci-
mens containing endotoxin by the formula antilog Ze/f, in which
Ze is the sum of the log end-points of the dilution series used and

? For a test for validity of procedure for inactivation of endotoxins,
see “Dry-heat Sterilization” under Sterilization and Sterility As-
surance of Compendial Articles (1211). Use a LAL Reagent
having a sensitivity of not less than 0.15 Endotoxin Unit per mL.


Toxaglate Sodium

Iron Dextran Injection

Iron Sorbitex

Isobucaine HC! and Epinephrine
Isoniazid

Isoproterenol HCI Injection
Isosulfan Sulfate

Isoxsupine HCI Injection

Kanamycin Sulfate Injection
Ketamine HCI

Labetalol HCI

Leucovorin Calcium Injection
Leuprolide Acetate

Levallorphan Tartrate Injection
Levarterenol

Levorphanol Tartrate Injection
Levothyroxine Sodium for Injection
Lidocaine HCI Injection (with D5W)
Lidocaine HCI with Epinephrine
Lincomycin HCI

Liver Derivative Complex
Lorazepam

Loxapine

Magnesium Sulfate

Manganese Chloride Injection
Manganese Sulfate

Mannitol <= 10%

Mannitol > 10%

Mannitol and Sodium Chloride
Mechlorethamine HCI for Injection
Medroxyprogesterone Acetate
Menadiol Sodium Diphosphate (K-4)

3.00 mL
0.90 mg
0.50 mL
0.14 mL
20.00 mg
0.014 mg
0.71 mg
0.14 mg

-K-

7.50 mg
13.00 mg

-L-

4.30 mg
2.57 mg
0.03 mg
0.04 mg
0.06 mg
0.04 mg
0.007 mg
4.50 mg
7.00 mg
10.00 mg
0.03 mg
0.05 mg
0.71 mg

-M-

57.10 mg
11.00 ug
11.00 ug
120.00 mg
2.00 g
120.00 mg
0.40 mg
14.30 mg
0.20 mg

45

1.67
5.60
10.00 +
35.70

0.30 +
350.00

7.00
35.70 +

0.67 +
0.40 +

1.20 +
1.95
16.67
125.00
83.33
125.00 +
714.00
1.10 +
0.70 +
0.50 +
166.67
100.00 +
7.00

0.09 +
0.45 +
0.45 +
0.04
2.50
0.04 +
12.50 #+
0.35
25.00 +


INTRODUCTION

This guideline sets forth acceptable conditions for use of the
Limulus Amebocyte Lysate test. It also describes procedures for
using this methodology as an end-product endotoxin test for human
injectable drugs (including biological products), animal injectable
drugs, and medical devices. The procedures may be used in lieu of

the rabbit pyrogen test.

For the purpose of this guideline, the terms “lysate” or "lysate
reagent" refer only to Limulus Amebocyte Lysate licensed by the
Center for Biologic Evaluation and Research. The term “official
test" means that a test is referenced in a United States
Pharmacopeia drug monograph, a New Drug Application, New Animal Drug

Application or a Biological License.


Cefoxitin Sodium

Ceftazidime

Ceftizoxime Sodium

Ceftriaxone Sodium

Cefuroxime Sodium

Cephacetrile Sodium for Injection

Cephaloridine

Cephalothin Sodium Injection

Cephapirin Sodium

Cephradine for Injection

Cerulitide diethylamine

Chloramphenicol Sodium Succinate

Chlordiazepoxide HCI

Chloroprocaine HCl

Cholecystokinin

Chorionic Gonadotropin

Chlormerodrin Hg197 Injection

Chlormerodrin Hg203 Injection

Chlormerodrine

Chloroquine HCI Injection

Chloroprocaine HCI

Chloroprocaine HCI - Epinephrine

Chlorothiazide Sodium

Chlorpheniramine Maleate

Chlorpromazine HCl

Chlorprothixene Injection

Chlortetracycline HCI

Chromate Sodium Cr51 Injection

Chromic Chloride Injection

Chromic Phosphate P32 Suspension

Chymopapain

Chymotrypsin

Sterile Cilastatin Sodium

Cimetidine HCI Injection

Cisplatin for Injection

Citric Acid, Magnesium Oxide,
& Sodium Carbonate Irrigation

Citrate, Phosphate, Dextrose, Adenine

Clindamycin Phosphate Injection

Cloxacillin

Codeine Phosphate Injection

40.00 mg 0.13 +
50.00 mg 0.10 +
50.00 mg 0.10 +
28.60 mg 0.20 +
50.00 mg 0.10 +
80.00 mg 0.06
14.30 mg 0.35
60.00 mg 0.08 +
28.60 mg 0.17 +
25.00 mg 0.20 +
0.30 mcg 16.67
25.00 mg 0.20 +
1.40 mg 3.57 +
11.43 mg 0.45
1.00 IDU 5.00
142.90 units 0.03 +
7.00 mL 25.00 +
7.00 mL 25.00 +
1.40 mg 3.57
7.50 mg 0.70 +
20.00 mg 0.25
20.00 mg 0.25
15.00 mg 0.30 +
0.57 mg 8.80 +
0.72 mg 6.90 +
0.72 mg 6.90 +
5.00 mg 1.00 +
7.00 mL 25.00 +
0.30 ug 16.70
7.00 mL 25.00 +
42.90 pKat 0.12
4.30 units 1.16
-.-- mg 0.23 +
10.00 mg 0.50
2.57 mg 1.90 #
-.-- mL 0.50 +
0.90 mL 5.56
8.60 mg 0.58 +
12.50 mg 0.40 +
0.86 mg 5.80 +

38


curve each day if consistency of etandard curves is shown in your test
laboratory. Datermine consistency by regression analysis of the data points from
the standard curvea generated over three consecutive test days (minimum of three
Curves). If the coefficient of correlation, r, meets the criteria in Appendix
A then consistency is proven and the curve becomes the “archived curve." If r
does not meet the criteria then consistency in your laboratory has not been shown
and you cannot use an archived curve in routine testing. The archived curve is
only valid for a lysate/endotoxin lot combination. If you use an archived
standard curve , at least duplicates of a standard endotoxin concentration,
equal to the mid-point on a log basis, between the endotoxin concentration of the
highest and lowest standards in the standard curve, in water must be included
with each run of samples. The mean endotoxin concentration of thie standard
control must be within Plus/minue 25% of the standard curve concentration when
calculated using the archived standard curve. Independent of using an endotoxin
standard curve, at least duplicates of a standard endotoxin in each product or
Product dilution (positive Product control), equal to either 0.1 - 0.5 or 1.0 ~
$.0 EU/mL depending on its PFC or 4 lambda, must be included with each run of
samples. The mean endotoxin concentration of the positive Product control when
referenced to the standard curve must be within plus/ minua SO®% of the known

concentration after subtraction of any endogenous endotoxin. An endotoxin
standard series should be run when retesting to determine if end-product
endotoxin contamination exceeds product Limit. If you use the alternate

Procedure, a standard curve prepared in product must Se conducted with each
Product tea..

APPENDIX A

Using a RSE or CSE of known potency, in endotoxin units, aseay at least 3
concentrations in triplicate that extend over the desired endotoxin range.
Additional standards should be included to bracket each log increase in the range
of the standard curve so that their is at least one standard per log increment
of the range. Do regression - correlation analysis on the log Reaction Time

The coefficient of correlation, r, shall be greater than or equal to the absolute
value of 0.980. If r is less than the absolute value of 0.980 the cause of the
non-linearity should be determined and test repeated.


EXAMPLE:

RSE Standard Curve

Concentration O.D.

RSE (EU/mL) o.1 0.11
0.25 0.26

0.5 0.49

1.0 1.06

y-intercept = —0.008 slope = 1.056 r = 0.999

Straight Line Equation (Y) = -0.008 + (1.056 * X)

CSE Standard Curve

CSE AVERAGE 0.D. Corresponding EU/ng
Conc. (ng/mL) RSE (EU/mL) (RSE/CSE)
0.01 0.12 0.119 11.9
0.025 0.31 0.301 12.0
0.05 0.60 0.626 12.5
0.1 1.23 1,291 12.9

Mean EU/ng = 12.3

CG. KINETIC-TURBIDIMETRIC TECHNIQUE

In order to assign EUs to a CSE, the following should be performed
on 4 vials from the same CSE lot.

Twofold dilutions of the RSE should be made in the range of 1.0
EU/mL to 0.03 EU/mL. Determine the Time of Reaction (T) for at
least duplicates of each standard concentration. Construct a
standard curve (Logjyg T versus Logjo endotoxin concentration
(E)). Calculate the mean T for 1.0 and 0.03 EU/mL. These T's
define the RSE standard range.

For each of the four vials of CSE make twofold dilutions such that
the T values for at least 3 concentrations of the CSE are within
the RSE standard range. Determine the T values for at least
duplicates of each endotoxin concentration. Calculate the EU/ng of
CSE by inserting the log mean CSE T values for each endotoxin
concentration which falls in the RSE standard range into the RSE
straight line equation. The resulting CSE values (in EU) are then
divided by their corresponding concentrations (in ng/mL). These
values are averaged to obtain the potency of the CSE lot.

~ 20 -


APPENDIX B

BACTERIAL ENDOTOXINS TEST

United States Pharmacopeia XXI/National Formulary XVI
and
First Supplement to USP XXI/NF XVI


Colchicine Injection
Colistimethate Sodium
Conjugated Estrogens
Corticotropin, Gel, Zinc & Re.
Cortisone Acetate

Cosyntropin

Cryptenamine Acetate

Cupric Chloride Injection
Cupric Sulfate Injection
Cyanocobalamine and Repository
Cyclizine Lactate
Cyclophosphamide

Cyclosporine Injection and Conc.
Cysteine HCI

Cytarabine

*Cytarabine

Dacarbazine for Injection
Dactinomycin for Injection
Dantrolene Sodium

Daunorubicin HCl
Decamethonium Bromide
Deferoxamine Mesylate
Dehydrocholate Sodium Injection
Deslanoside

Desmopressin Acetate
Desoxycorticosterone Acetate
Desoxycorticosterone Pivalate Sus.
Dexamethasone Acetate Suspension
Dexamethasone Sodium Phosphate
Dexpanthenol

Dextran 40

Dextran 40 in Sodium Chloride
Dextran 70

Dextrose <5%

Dextrose- 5%-70%

Dextrose and Sodium Chloride
Dezocine

0.03 mg 166.70 +
2.50 mg 2.00 +
0.36 mg 13.89
1.60 units 3.10 +
5.00 mg 1.00
3.57 mcg 1.40
1.86 CSR units 2.69
0.02 mg 250.00 +
0.02 mg 250.00 +
14.30 meg 0.40 +
1.00 mg 5.00
30.00 mg 0.20 #+
0.12 mL 42.00 +
7.14 mg 0.70 +
3.00 mg 0.07 +
1.93 mg 0.10 #
-D-
9.60 mg 0.52 #
0.05 mg 100.00 #+
10.00 mg 0.50
1.16 mg 4.30 #
0.043 mg 116.30
15.00 mg 0.33
150.00 mg 0.04
0.03 mg 167.00
0.30 mcg 16.70
0.07 mg 71.40 +
1.80 mg 2.78 +
0.23 mg 21.74 +
0.16 mg 31.30 +
7.10 mg 0.70
5.00 mL 1.00
5.00 mL 1.00
10.00 mL 0.50
10.00 mL 0.50
0.50 gm 10.00
0.50 gm 10.00
0.29 mg 17.24

39


The

b. For unusually small or large devices, the surface area
of the device which comes in contact with the patient
may be used as an adjustment factor in selecting the
rinsing or extracting volume. The endotoxin limit can
be adjusted accordingly.

rinsing scheme should not result in a greater dilution of

endotoxin than used in USP rabbit pyrogen testing of transfusion

and

infusion assemblies. For inhibition/enhancement testing,

both the rinsing/extraction solution and the device
eluate/extract should be tested as prescribed below under the
specific technique being used.

a) GEL-CLOT TECHNIQUE

b)

In inhibition/enhancement testing, a device eluate/extract
containing varying concentrations of a standard endotoxin
that bracket the sensitivity of the lysate is compared with
a series of the same endotoxin concentrations in water
alone. The device eluate/extract is "spiked" with endotoxin
and then diluted with additional’ eluate/extract to the same
endotoxin concentrations as in the water series. Results of
endotoxin determination in water and the device product
eluate/extract should fall within plus/minus a twofold

dilution of the labeled sensitivity. If the device
eluate/extract shows inhibition, the gel-clot technique
cannot be used to test the device. Negative controls

(diluent plus lysate) should be included in all
inhibition/enhancement testing.

CHROMOGENIC AND ENDPOINT-TURBIDIMETRIC TECHNIQUES

In inhibition/enhancement testing by these techniques, a@
device eluate/extract containing 4 lambda concentration of
the RSE or CSE (lambda is equal to the lowest endotoxin
concentration used to generate the standard curve) is tested
in duplicate according to the lysate manufacturer's
methodology. The standard curve for these techniques shall
consist of at least four RSE or CSE concentrations in water
that extend over the desired range. If the desired range is
greater than one log, additional standard concentrations
should be included. The standard curve must meet the
criteria for linearity as outlined in Appendix A(2). The
detected amount of endotoxin in the spiked eluate/extract
must be within plus or minus 25% of the 4 lambda
concentration for the device to be considered to neither
enhance nor inhibit the assay. If the device eluate/extract
shows inhibition, the device cannot be tested by this
technique.

An alternate procedure may be performed as described above

except the RSE/CSE standard curve is Prepared in LAL

negative device eluate/extract, i.e. no detectable

endotoxin, instead of LAL negative water. The standard

curve must meet the test for linearity, i.e. r equal to or

greater than 0.980, and in addition the difference between
-13-


when validated according to this guideline, is at least
equivalent to the rabbit pyrogen test as an end-product test for
medical devices. A manufacturer labeling a device as
non-pyrogenic must validate the LAL test for that device in the
test laboratory to be used for end-product testing before using
the LAL test as an end-product endotoxin test for any device.

The data discussed under Section V of this guideline may be
expressed graphically or in tabular form and should be on file
at the manufacturing site; no preclearance prior to use of the
LAL test as an end-product test is required if it is used
according to this FDA guideline. Voluntary submission of LAL
validation and inhibition data obtained following issuance of
this guideline will be accepted for CDRH review and comment.

When a manufacturer plans to use LAL test procedures that
deviate significantly from the LAL guideline, a premarket
notification under section 510(k) of the Federal Food, Drug, and
Cosmetic Act (the Act) or a Premarket Approval Application (PMA)
supplement under section 515 of the Act should be submitted.
Significant deviations would -include-— but not necessarily be
limited to-- higher endotoxin concentration release criteria,
sampling from fewer than three lots for inhibition/enhancement
testing, lesser sensitivity to endotoxin, rabbit retest when the
LAL method shows endotoxin above the recommended allowable
endotoxin dose, and a device rinsing protocol resulting in
greater dilution of endotoxin than that recommended in this

guideline.

CDRH will also consider submissions in the form of a premarket
notification or PMA supplement for another deviation from this
draft guideline; process control of endotoxin contamination with
reduced end-product testing, i.e., a decrease in the number of
devices per lot undergoing end-product testing. The
manufacturer must demonstrate adequate control of the production
Process by the use of routine checks for endotoxin at key stages
of production except where it has been shown that no possibility
of contamination exists.

To facilitate subsequent PMA review, providers of
investigational devices subject to 21 CFR part 812 or 813 are
encouraged to use this guideline when a non-pyrogenic device is
to be manufactured.


Amphotericin B for Injection

*Amphotericin B for Injection

Ampicillin Sodium

Ampicillin and Sulbactam

Amrinone Lactate

Anileridine

Anticoagulant Heparin Solution

Anticoagulant, Citrate Dextrose Sol.

Anticoagulant, Citrate Phosphate Dextrose

Anticoagulant, Citrate Phosphate Dextrose
Adenine Solution

Antihemophilic Factor

Antihemophilic Plasma(1 hr. at 56-570C)

Antirabies Serum

Antitoxin (Gas Gangrene)

Antivenom

Apommorphine HCI Tablets for Injection

Arginine HCI Injection

Ascorbic Acid

Asparaginase for Injection

Atracurium Besylate

Atropine Sulfate

Aurothioglucose Suspension

Azathioprine Sodium for Injection

Azlocillin

Aztreonam for Injection

Bacitracin

Bacitracin Zinc

Benzquinamide HCI

Benztropine Mesylate Injection
Benzylpenicilloy! Polylysine
Betamethasone Acetate and
Betamethasone Sodium Phosphate
Betamethasone Sodium Phosphate
Betazole HCI Injection
Bethanechol Chloride

Biperiden Lactate Injection

1.00 mg
0.01 mg
33.30 mg
28.60 mg
10.00 mg
0.70 mg
2.00 mL

-.-- mL

-.-- mL
10.00 units
3.00 mL
3.00 mL
3.00 mL
3.00 mL
0.09 mg
500.00 mg
4.20 mg
1000.00 TU
0.50 mg
0.09 mg
0.70 mg
5.00 mg
75.00 mg
28.60 mg

-B-

500.00 units
500.00 units
1.00 mg
0.09 mg

0.004 mL

0.17 mg
0.17 mg
2.86 mg
0.20 mg
0.06 mg

36

5.00
20.00
0.15 +
0.17
0.50
7.20 +
2.50
5.56

5.56

5.56
0.50
1.67
1.67
1.67
1.67
55.56
0.01
1.20 +
0.01 #
10.00
55.60 +
7.14
1.00 +
0.07 +
0.17 +

0.01 +

0.01

5.00
55.60 +

1250.00

29.20 +
29.20 +
1.75

25.00
83.30 +


APPENDIX D (cont.)

Step 2. Maximum Valid Dilution (MVD)

For drugs administered on a weight-per-kilogram basis, the
Potency is expressed as mg or units/mL and for drugs
administered on a volume-per-kilogram, the potency is equal to
1.0 mL/mL.

METHOD I EXAMPLES

Endotoxin Limit Expressed by Weight:

Product: Cyclophosphamide Injection
Potency: 20 mg/mL

Lysate Sensitivity (A): 0.065 EU/mL
Endotoxin Limit (Appendix E): 0.17 EU/mg

MvD = —0.17 EU/mg X_20 mg/ml _ _ 3.4 = 1:52.3 or 1:52
0.065 EU/mL 0.065

Endotoxin Limit Expressed by Volume:

Product: 5% Dextrose Injection
Lysate Sensitivity (A): 0.065 EU/mL
Endotoxin Limit (Appendix E): 0.5 EU/mL,

mvp = 0.5 EU/mL X 1 mb/mb = 0.5 _ 4, 7.7

0.065 EU/mL 0.065

METHOD II EXAMPLES

PARENTERAL DRUGS EXCEPT INTRATHECAL

Drug Administered on a Weight-per-Kilogram Basis

Product: Cyclophosphamide Injection
Potency: 20 mg/mL

Maximum Dose/Kg ( M ): 30 mg/Kg
Lysate Sensitivity (2): 0.065 EU/mL

mvc = A.M  _ 0.065 EU/mL X 30 mg/Kg _ 0.390 mg/mL

K 5.0 EU/Kg

Mvp = Potency of Product - _20 mg/mL _ 1:51.2 or 1:51
MVC 0.390 mg/mL

~ 23 -


available to interested parties to permit manufacturers, especially those
who had used the LAL test in parallel with the rabbit pyrogen test, to
submit data that could be considered in the preparation of any final
guideline.

In response to comments received on the January 18 draft guideline, FDA
made several significant changes (i.e. Endotoxin limits changed and
deletion of section on Absence of Non-endotoxin Pyrogenic Substances),
and many minor editorial changes. The agency also determined that a
single document should be made available covering all FDA regulated
products that may be subject to LAL testing. Primarily because of the
addition of biological products and medical devices to the guideline, the
the agency made, in the FEDERAL REGISTER of March 29, 1983 (43 FR 13096),
another draft of the guideline available for public comment.

Based on the comments received on the March 29 draft guideline, FDA has
made several changes in this final guideline. The comments used in
support of these changes may be viewed at FDA's Dockets Management
Branch, Room 4-62, 5600 Fishers Lane, Rockville, MD between 9 am and 4 pm
Monday through Friday. Briefly, the significant changes made are:

A. Inclusion of validation criteria for the chromogenic,
endpoint-turbidimetric and kinetic-turbidimetric LAL techniques.

B. Any technique (gel-clot, chromogenic or turbidimetric) can be
used in testing a product for endotoxin. However, if a gel-clot
lysate is used in a different technique the results must be
interpreted using the criteria for the technique being used.

C. Elimination of the requirement to test the sensitivity of a
rabbit pyrogen testing colony.

D. The Center for Devices and Radiological Health (CDRH) has
adopted the USP Endotoxin Reference Standard and revised the
limit expressions from ng/mL to EU/mL. The new limit for
medical devices is 0.5 EU/mL except for devices in contact with
cerebrospinal fluid for which the limit is 0.06 EU/mL. These
limits for devices are equivalent to those for drugs for a 70 Kg
man when consideration is given to the following:

1. In the worst case situation, all endotoxin present in
the combined rinsings of 10 devices could have come from
just one device. A wide variation in bioburden is
common to some devices.

2. Published FDA studies indicate that less than half of
added endotoxin is recovered from devices using a
non-pyrogenic water rinse.

E. The Center for Drug Evaluation and Research (CDER) has added a
listing of the maximum doses per Kg per hour and the
corresponding endotoxin limits for most of the aqueous
injectable drugs and biologics currently on the market. This
listing was added to promote uniformity among companies making
the same product.


I. BACKGROUND

In a notice of January 12, 1973 (38 FR 1404), FDA announced that Limulus
Amebocyte Lysate (LAL), derived from circulating blood cells
(amebocytes) of the horseshoe crab, (Limulus polyphemus), is a biological
Product. As such, it is subject to licensing requirements as provided in
section 351 of the Public Health Service Act (42 U.S.C. 262). Since
1973, LAL has proved to be a sensitive indicator of the presence of
bacterial endotoxins (pyrogens). Because of this demonstrated
sensitivity, LAL can be of value in preventing the administration or use
of products which may produce fever, shock, and death if administered to
or used in humans or animals when bacterial endotoxins are present.

When the January 12, 1973 notice was published, available data and
experience with LAL were not adequate to support its adoption as the
final pyrogen test in place of the rabbit pyrogen test, which had been
accepted and recognized for many years. In order to establish a data
base and gain experience with the use of LAL, that notice permitted the
introduction of LAL into the marketplace without a license. This was
upon the condition that its use be limited to the in-process testing of
drugs and other products, that the decision to use it be reached
voluntarily by affected firms, and the labeling on LAL state that the
test was not suitable as a replacement for the rabbit pyrogen test.

Since that time, production techniques have been greatly improved and
standardized so that they consistently yield LAL with an endotoxin
sensitivity over 100 times greater than originally obtained. Moreover,
it is widely recognized that the LAL test is faster, more economical, and
requires a smaller volume of Product than does the rabbit pyrogen test.
In addition, the procedure is less labor intensive than the rabbit test,
making it possible to perform many tests in a single day.

In a notice published in the Federal Register of November 4, 1977 (42 FR
57749), FDA described conditions for the use of LAL as an end-product
test for endotoxins in human biological products and medical devices.
The notice stated further that the application of LAL testing to human
drug products would be the subject of a future Federal Register
publication.

The then Bureau of Medical Devices, now FDA's Center for Devices and
Radiologic Health (CDRH), issued recommended procedures for the use of
LAL testing as an end-product endotoxin test on March 26, 1979. These
procedures were revised as a result of the comments received from
interested parties .

As a direct result of CDRH's experience in approving petitions for the
use of the LAL test in place of the rabbit pyrogen test, several
Procedures for using the LAL test have evolved and have been adopted for
devices.

In the FEDERAL REGISTER of January 18, 1980 (45 FR 3668), FDA announced
the availability of a draft guideline that set forth procedures for use
of the LAL test as an end-product testing method for endotoxins in human
and animal injectable drug products. This draft guideline was made

~.le


the O.D. readings for the lowest and highest endotoxin
concentrations must be greater than 0.4 and less than 1.5
O.D. units, If the standard curve does not meet these
criteria the device cannot be tested by the alternate
procedure.


III. REGULATORY PROVISIONS THAT PERMIT INITIATION
OF END-PRODUCT TESTING WITH LAL

The regulatory provisions that a firm must meet before using the LAL test
as an end-product test are not the same for all categories of products

because

of the different applicable statutory provisions and

regulations. These provisions are as follows:

A.

Human Drugs subject to New Drug Applications (NDAs) or
Abbreviated New Drug Applications (ANDAs) Antibiotic Drug
Applications and animal drugs subject to New Animal Drug
Applications (NADAS) and Abbreviated New _ Animal Drug

Application.

For these classes of drugs, manufacturers are to submit a
supplemental application to provide for LAL testing. However,
under 21 CFR 314.70(c) for drugs for human use and 21 CFR
514.8(d)(3) for drugs for animal use various changes may be made
before FDA approval. Under these sections changes in testing of
a human or animal drug that give increased assurance that the
drug will have the characteristics of purity it purports or is
represented to possess should be placed into effect at the
earliest possible time. Therefore, if a firm validates the LAL
test for a particular drug product covered by a new drug
application by the procedures in this guideline using a LAL
reagent licensed by the Center for Biologic Evaluation and
Research (OBER) for the technique being used, the change may be
made concurrently with the submission of the supplement
providing for it. The supplement should contain initial quality
control data, inhibition/enhancement data and the endotoxin
limit for the drug product.

Biological products for human use.

Under 21 CFR 601.12 significant changes in the manufacturing
methods of biological products are required to be reported to
the agency and may not become effective until approved by the
Director, OBER. Therefore, a manufacturer of a biological
product shall obtain an approved amendment to its product
license before changing to the use of LAL in an end-product
test, irrespective of the validation procedure used.

Drugs not subject to premarket approval.

A manufacturer of an injectable drug for human or animal use
that is not subject to premarket approval would be able to use
the LAL test as an end-product test for endotoxins without
submitting any information to the agency. CGMPs require the
manufacturer to have data on file to validate the use of the LAL
test for each product for which it is being used.

Medical Devices.

On the basis of extensive experience in review of LAL data on
devices since November 1977, CDRH believes that the LAL test,
-4-


Sis the number of replicate end-points in cach case. Compute the
amount of endotoxin in the specimen to which endotoxin has been
added. The test is valid for the article if this result is within twofold
of the known added amount of endotoxin. Alternatively, if the test
has been appropriately set up, the test is valid for the article if the
geometric mean end-point dilution for the specimen to which en-
dotoxin has been added is within one 2-fold dilution of the corre-
sponding geometric mean end-point dilution of the standard en-
dotoxin.

If the result obtained for the specimens to which endotoxin has
been added is outside the specified limit, the articic is unsuitable
for the Bacterial Endotoxins Test, or, in the case of Injections or
solutions for parenteral administration, it may be rendered suitable
by diluting specimens appropriately.

Repeat the test for inhibition or enhancement using specimens
diluted by a factor not exceeding that given by the formula x/A (see
Maximum Valid Dilution, below). Use the least dilution sufficient
to overcome the inhibition or enhancement of the known added
endotoxin, for subsequent assays of endotoxin in test specimens.

If endogencous endotoxin is detectable in the untreated specimens
under the conditions of the test, the article is unsuitable for the
Inhibition or Enhancement Test, or, it may be rendered suitable
by removing the endotoxin present by ultra-filtration, or by ap-
propriate dilution. Dilute the untreated specimen (as constituted,
where applicable, for administration or use), toa level not exceeding

the maximum valid dilution, at which no endotoxin is detectable.
"4, Repeat the test for Inhibition or Enhancement using the
specimens at those dilutions.

Test Procedure

In preparing for and applying the test, observe precautions in
handling the specimens in order to avoid gross microbial contami-
nation. Washings or rinsings of devices must be with LAL Reagent
Water in volumes appropriate to their use and, where applicable,
of the surface area which comes into contact with body tissues or
fluids. Use such washings or rinsings if the extracting fluid has been
in contact with the relevant pathway or surface for not less than |
hour at controlled room temperature (15° to "30° m1). Such ex-
tracts may be combined, where appropriate. The ultimate rinse
or wash volume is such as to result in possible dilution of any con-
tained endotoxin to a level not less than that suitable for use in the
Pyrogen Test (151) under Transfusion and Infusion Assemblies
(161).

For validating the test for an article, for endotoxin limit tests or
assays, or for special purposes where so specified, testing of speci-
mens is conducted quantitatively to determine response end-points
for gel-clot readings. Usually graded strengths of the specimen and
standard endotoxin are made by multifold dilutions. "Select
dilutions; so that they correspond to a geometric series in which
cach step is greater than the next lower by a constant ratio."—; Do
not store diluted endotoxin, because of loss of activity by adsorption.
In the absence of supporting data to the contrary, negative and
positive controls are incorporated in the test.

Use not less than 2 replicate reaction tubes at each level of the
dilution series for each specimen under test. Whether the test is
employed as a limit test or as a quantitative assay, a standard en-
dotoxin dilution series involving not less than 2 replicate reaction
tubes is conducted in parallel. A set of standard endotoxin dilution
series is included for each block of tubes, which may consist of a
number of racks for incubation together. "gy provided the envi-
ronmental conditions within blocks are uniform.

Preparation—Since the form and amount per container of

standard endotoxin and of LAL reagent may vary, constitution
and/or dilution of contents should be as directed in the labeling. "a1
The pH of the test mixture of the specimen and the LAL Reagent
is in the range 6.0 to 7.5 unless specifically directed otherwise in the
individual monograph. The pH may be adjusted by the addition
‘of sterile, endotoxin-free sodium hydroxide or hydrochloric acid or
suitable buffers to the specimen prior to testing.

Maximum Valid Dilution (MYD)—The Maximum Valid
Dilution is appropriate 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 thé dosage form for administration could be varied. "Where the
endotoxin limit concentration is specified in the individual mono-
graph in terms of volume (in EU per mL), divide the limit by A.
which is the labeled sensitivity (in EU per mL) of the lysate em-
ployed in the assay, to obtain the MVD factor. Where the endo-
toxin limit concentration is specified in the individual monograph
in terms of weight or of 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 appli-
cable, and divide the product of the multiplication by A, to obtain
the MVD factor. The MVD factor so obtainede; is the limit
dilution factor for the preparation for the test to be valid.

Procedure—To 10- X 75-mm test.tubes add aliquots of the ap-
propriately constituted LAL reagent, and the specified volumes of
specimens, endotoxin standard, negative controls, and a positive
product control consisting of the article, or of solutions, washings
or extracts thereof to which the RSE (or a standardized CSE) has
been added at a concentration of endotoxin of 2A for that LAL re-
agent (see under Test for confirmation of labeled LAL Reagent
sensitivity). Swirl each gently to mix, and place in an incubating
device such as a water bath or heating block, accurately recording
the time at which the tubes are so placed. Incubate cach tube,
undisturbed, for 60 + 2 minutes at 37 + 1°, and carefully remove
it for observation. A positive reaction is characterized by the for-
mation of a firm gel that remains when inverted through 180°.
Record such a result as positive (+). A negative result is charac-
terized by the absence of such a gel or by the formation of a viscous
gel that does not maintain its integrity. Record such a result as
negative (—). Handle the tubes with care, and avoid subjecting
them to unwanted vibrations, or false negative observations may
result. The test is invalid if the positive product control or the en-
dotoxin standard does not show the end-point concentration to be
within +1 twofold dilutions from the label claim sensitivity of the
LAL Reagent or if any negative control shows a gel-clot end-

point.

Calculation and Interpretation

Calculation—Calculate the concentration of endotoxin (in Units
per mL or in Units per g or mg) in or on the article under test by the
formula pS/U. in which S is the antilog of the geometric mean logio
of the end-points, expressed in Endotoxin Units (EU) per mL for
the Standard Endotoxin. U is the antilog of De/f, where e is the
logo of the end-point dilution factors, expressed in decimal frac-
tons, f is the number of replicate reaction tubes read at the end-
point level for the specimen under test. and p is the correction factor
for those cases where a specimen of the article cannot be taken di-
rectly into test but is processed as an extract, solution, or
washing

Where the test is conducted as an assay with sufficient replication
to provide u suitable number of independent results, calculate for
¢ach replicate assay the concentration of endotoxin in or on the
article under test from the antilog of the geometric mean log end-
point ratios. Calculate the mean and the confidence limits from
the replicate logarithmic values of all the obtained assay results by
a suitable statistical method (see Calculation of Potency from a

Single Assay (111))

Interpretation —The article mects the requirements of the test
if the concentration of endotoxin does not exceed that specified in
the individual monograph. and where so specified in the individual
monograph or in this chapter, the confidence limits of the assay do
not exceed those specified


CENTER For Druc EVALUATION AND RESEARCH

Guidance for Industry

The FDA published Good Guidance Practices in February 1997.
This guidance was developed and issued prior to that date.

Additional copies are available from:
Office of Training and Communications
Division of Communications Management
Drug Information Branch, HFD-210
5600 Fishers Lane
Rockville, MD 20857

(Tel) 301-827-4573
(Internet) http://www.fda. gov/cder/guidance/index.htm

U.S. DeparTMENT OF HEALTH AND Human Services, Foop AND DruG ADMINISTRATION



Sodium Bicarbonate

Sodium Chloride 0.45-0.9%
Sodium Chloride 3- 24.3%

Sodium Chloride - Bacteriostatic
Sodium Chloride 4.5%- Lactose 3%
Sodium Chloride Irrigation
Sodium Citrate

Sodium Iodide

Sodium Lactate

Sodium Phosphate Injection
Sodium Phosphate P32 Solution
Sodium Salicylate

Sodium Tetradecyl Sulfate

Sodium Thiosalicylate

Sodium Thiosulfate

Somatrem for Injection
Somatropin - Pituitary & Recombinant
Soybean Oil Emulsion
Spectinomycin HCl

Streptokinase
Streptokinase-Streptodornase (Local)
Streptokinase-Streptodornase (IM)
Streptomycin Sulfate

Streptozocin

Succinylcholine Chloride
Sufentanil citrate

Invert Sugar

Invert Sugar in Sodium Chloride
Sulbactam Sodium

Sulfadiazine Sodium
Sulfamethoxazole & Trimethoprim
Sulfisoxazole Diolamine Injection
Sulfobromophthalein

Technetium Tc99m Albumin Aggregated
Technetium Tc99m Antimony Trisulfate
Technetium Tc99m Dsofenin
Technetium Tc99m Etidronate
Technetium Tc99m Ferpentetate

1.00 mEq 5.00 +
10.00 mL 0.50 +
1.40 mL 3.57 +
5.00 mL 1.00 +
10.00 mL 0.50
10.00 mL 0.50 +
2.50 mEq 2.00
14.30 mg 0.35
2.40 mEq 2.00 +
4.00 mg 1.10 +
7.00 mL 25.00 +
9.30 mg 0.54
0.14 mL 35.71
2.10 mg 2.38
167.00 mg 0.03 +
0.25 IU 20.00
0.20 IU 25.00
3.13 mL 1.60
57.00 mg 0.09 +
21428.00 IU 0.02/100
3000.00 units 0.002
1000.00 units 0.005
20.00 mg 0.25 +
38.60 mg 0.13 #
2.50 mg 2.00 +
0.05 mg 100.00
10.00 mL 0.50 +
10.00 mL 0.50
14.30 mg 0.35
50.00 mg 0.10 +
25.00 mg(sulf) 0.20
50.00 mg 0.10
5.00 mg 1.00 +
-T-
7.00 mL 25.00 +
7.00 mL 25.00
7.00 mL 25.00 +
7.00 mL 25.00 +
7.00 mL 25.00 +


b)

water, Results of endotoxin determination in water and the
drug product should fall within plus/minus a_ twofold
dilution of the labeled sensitivity. If the undiluted drug
product shows inhibition, the drug product can be diluted,
not to exceed the MVD, with the same diluent that will be
used in the release testing and the above procedure
repeated. Negative controls (diluent plus lysate) should be
included in all inhibition/enhancement testing.

CHROMOGENIC AND ENDPOINT-TURBIDIMETRIC TECHNIQUES

In inhibition/enhancement testing by these techniques, a
drug concentration containing 4 lambda concentration of the
RSE or CSE (lambda is equal to the lowest endotoxin
concentration used to generate the standard curve) is tested
in duplicate according to the lysate manufacturer's
methodology. The standard curve for these techniques shall
consist of at least four RSE or CSE concentrations in water
that extend over the desired range. If the desired range is
greater than one log, additional standards concentrations
should be included. The standard curve must meet the
criteria for linearity as outlined in Appendix A(2). The
detected amount of endotoxin in the spiked drug must be
within plus or minus 25% of the 4 lambda concentration for
the drug concentration to be considered to neither enhance
nor inhibit the assay. If the undiluted drug product shows
inhibition, the drug product can be diluted, not to exceed
the MVD, and th test repeated.

An alternate procedure may be performed as described above
except the RSE/CSE standard curve is prepared in LAL
negative drug product, i.e. no detectable endotoxin, instead
of LAL negative water. The standard curve must meet the
test for linearity, i.e. r equal to or greater than 0.980,
and in addition the difference between the 0.D. readings for
the lowest and highest endotoxin concentrations must be
greater than 0.4 and less than 1.5 0O.D. units. If the
Standard curve does not meet these criteria, the drug
product cannot be tested by the alternate procedure.

c) KINETIC-TURBIDIMETRIC TECHNIQUE

In inhibition/enhancement testing by this technique, a drug
concentration containing 4 lambda concentration of the RSE
or CSE (lambda is equal to the lowest endotoxin
concentration used to generate the standard curve) is tested
in duplicate according to the lysate manufacturer's
methodology. The standard curve shall consist of at least
four RSE or CSE concentrations. If the desired range is
greater than one log, additional standard concentrations
should be included. The standard curve must meet the
criteria outlined in Appendix A(3). The calculated mean
amount of endotoxin in the spiked drug product, when
referenced to the standard curve, must be within plus or
minus 25% to be considered to neither enhance nor inhibit
the assay. If the undiluted drug product shows

7-


IV. HUMAN AND ANIMAL DRUGS AND BIOLOGICAL PRODUCTS

GENERAL REQUIREMENT

Manufacturers shall use an LAL reagent licensed by CBER in all
validation, in-process, and end-product LAL tests.

A.

VALIDATION QF THE LAL TEST

Validation of the LAL test as an endotoxin test for the release of

human and animal drugs includes the following: (1) initial

qualification of the laboratory, and (2) inhibition and enhancement

tests.

1. INITIAL QUALIFICATION OF THE LABORATORY
Various methodologies have been described for the detection of
endotoxin, using, limulus amebocyte lysate. Currently,
commercially available licensed lysates use the gel clot,
chromogenic, endpoint-turbidimetric or kinetic-turbidimetric
techniques. Other methods which have been reported show
potential for increasing further the sensitivity of the LAL
method,
Manufacturers should assess the variability of the testing
laboratory before any offical tests are performed. Each analyst
using a single lot of LAL and a single lot of endotoxin should
perform the test for confirmation of labeled LAL reagent
sensitivity or of performance criteria. Appendix A gives the
procedures and test criteria for the current licensed techniques.

2. INHIBITION AND ENHANCEMENT TESTING

The degree of product inhibition or enhancement of the LAL
procedure should be determined for each drug formulation before
the LAL test is used to assess the endotoxin content of any
drug. All validation tests should be performed on undiluted
drug product or on an appropriate dilution. Dilutions should
not exceed the Maximum Valid Dilution (MVD) (see Appendix D).
At least three production batches of each finished product
should be tested for inhibition and enhancement.

a) GEL-CLOT TECHNIQUE

Inhibition/enhancement testing should be conducted according
to the directions in the preparatory section of the USP
Bacterial Endotoxins Test (see Appendix B). Briefly, the
method involves taking a drug concentration containing
varying concentrations of a standard endotoxin that bracket
the sensitivity of the lysate and comparing it to a series
of the same endotoxin concentrations in water alone. The
drug product is “spiked" with endotoxin and then diluted
with additional drug product (so that the drug concentration
remains constant) to the same endotoxin concentrations in
~6-


Metoprolol Tartrate
*Metrizamide
Metrizamide
Metrizoic

Metronidazole HCI

Metyrapone Tartrate Injection

Mezlocillin Sodium
Miconazole Injection
*Miconazole Injection
Midazolam HCI
Minocycline HCI
Mithramycin for Injection
Mitomycin for Injection
Mitoxantrone HCI
Molybdenum

Morphine Sulfate
*Morphine Sulfate
Morrhuate Sodium
Moxalactam
Muromonab-CD3

Nafcillin

Nalbuphine HCI
Nalorphine HCl
Naloxone HCI Injection
Neomycin Sulfate
Neostigmine Methylsulfate
Netilmicin

Niacin

Niacinamide Injection
Nicotinamide
Nikethamide

Nine Vitamin Injection
Nitrofurantoin
Nitroglycerin
Nitroprusside Sodium
Norepinephrine bitartrate
Novobiocin for Injection

0.20 mg 25.00 +
4.29 mg I 1.17
634.00 mg I 0.008
73% - 2.90 mL 1.72
46.18% - 1.00 mL 5.00

15.00 mg 0.35 +

15.00 mg 0.35

87.50 mg 0.06 +

40.00 mg 0.10 +
0.29 mg 0.69
0.35 mg 14.30
4.00 mg 1.25 +
0.05 mg 100.00
0.51 mg 9.80 # +
0.36 mg 13.90 #
2.30 ug 2.17
0.29 mg 17.00
0.014 mg 14.29
3.60 mg 1.40 +

100.00 mg 0.05 +
0.10 mg 50.00
-N-

40.00 mg 0.13 +
3.00 mg 1.67
0.43 mg 11.60
0.01 mg 500.00
3.80 mg 1.30 +
0.04 mg 125.00
4.00 mg 1.25 +
1.43 mg 3.50 +
1.43 mg 3.50 +
0.70 mg 7.44
0.90 ml:25% sol 5.56
1.00 mL 5.00
2.50 mg 2.00

50.00 ug 0.10 +
1.40 meg 3.67
0.06 mg 83.40 +
7.10 mg 0.70

47


APPENDIX E

MAXIMUM DOSE AND ENDOTOXIN LIMIT TABLE

Drug Name

Acetic Acid Irrigation

Acetazolamide Sodium

Acetylcysteine Injection

Acyclovir Sodium

Adenosine Phosphate
Albumin,Normal Human Serum (25%)
Albumin,Normal Human Serum (20%)
Albumin,Normal Human Serum (5%)
Albuterol Sulfate

Alcohol and Dextrose Injection
Alfentanil Hydrochloride

Alkaloids of Belladonna
Alpha,-Proteinase Inhibitor
Alphaprodine HCI Injection
Alprostadil (Postaglandin)

Alteplase

Amdinocillin

Amikacin Sulfate Injection

Amino Acid Injection

Amino Acids and Electrolytes
Essential Amino Acids and Dextrose
Aminocaproic Acid Injection
Aminohippurate Sodium Injection
Aminophylline Injection
Amitriptyline HCI Injection
Ammonia N 13 Injection

Ammonium Chloride Injection
Amobarbital Sodium

Amoxicillin, Sterile and Suspension

Dose (M) Endotoxin Limit
(EU/mg, ml, units
of product)
-A-
10.00 mL 0.50 +
10.00 mg 0.50 +
150.00 mg 0.03
30.00 mg 0.17
0.71 mg 7.04
3.00 mL 1.67
3.75 mL 1.33
10.00 mL 0.50
0.008 mg 625.00
1.79 mL 2.70
250.00 mcg 0.02
0.007 mg 714.29
60.00 mg 0.08
0.60 mg 8.33
6.00 meg 0.83
1.25 mg 4.00
10.00 mg 0.50 +
15.00 mg 0.33 +
25.00 mg 0.20
25.00 mg 0.20
25.00 mg 0.20
100.00 mg 0.05
125.00 mg 0.04 +
5.00 mg 1.00
0.42 mg 12.00
7.00 mL 25.00 +
2.90 mEq Cl 1.72
14.30 mg 0.40 +
20.00 mg 0.25 +

35

April ,1992


Technetium Tc99m Gluceptate 7.00 mL
Technetium Tc99m Human Serum Albumin 7.00 mL

Technetium Tc99m Lidofenin 7.00 mL
Technetium Tc99m Mebrofenin 7.00 mL
Technetium Tc99m Medronate 7.00 mL
Technetium Tc99m Oxidronate 7.00 mL
Technetium Tc99m Pentetate 7.00 mL
Technetium Tc99m Sodium Pertechnetate 7.00 mL
Technetium Tc99m Pyrophosphate 7.00 mL
Technetium Tc99m (Pyro- and trimeta-)

Phosphates 7.00 mL
Technetium Tc99m Succimer 7.00 mL
Technetium Tc99m Sulfur Colloid 7.00 mL
Terbutaline Sulfate 0.004 mg
Teriparatide Acetate 5.00 units
Testolactone Suspension 1.43 mg
Testosterone (aqueous suspension) 1.43 mg
*Tetracaine Hydrochloride 0.29 mg
*Tetracaine HCI and Dextrose 0.20 mg
Tetracycline HCI 10.00 mg
Tetracycline Phosphate Complex 5.00 mg
Thallus Chloride T1201 Injection 7.00 mL
Theophylline and Dextrose 5.00 mg
Thiamine HCI 1.43 mg
Thiamylal Sodium 5.00 mg
Thiethylperazine Maleate 0.14 mg
Thiopental Sodium 5.00 mg
Thiotepa for Injection 0.80 mg
Thiothixene HCI Injection 0.057 mg
Thyrotropin for Injection 0.14 IU
Ticarcillin Disodium 100.00 mg
Ticarcillin Disodium and Clavulanate 75.00 mg
Tobramycin Sulfate 2.50 mg
Tolazoline HCI 6.00 mg
Tolbutamide Sodium 14.20 mg
Tranexamic Acid 10.00 mg
Triamcinolone Acetate Suspension 1.14 mg
Triamcinolone Acetonide 1.14 mg
Triamcinolone Diacetate Suspension 0.70 mg
Triamcinolone Hexacetonide Suspension 0.29 mg
Tridihexethyl Chloride 3.00 mg

52

25.00 +
25.00 +
25.00

25.00

25.00 +
25.00 +
25.00 +
25.00 +
25.00 +

25.00 +
25.00 +
25.00 +

1250.00 +

1.00
3.50
3.50 +
0.70 +
1.00 +
0.50 +
1.00 +
25.00 +
1.00
3.50
1.00 +
35.80 +
1.00 +
6.20 #
88.00 +
36.00
0.05 +
0.07 +
2.00 +
0.80 +
0.35 +
0.50
4.40 +
4.39
7.10 +
17.20 +
1.70 +


APPENDIX D (cont.)

Drug Administered on a Volume-per-Kilogram Basis

Product: 5% Dextrose in Water
Maximum Dose/Kg ( M ): 10.0 mL/Kg
Lysate Sensitivity (A): 0.065 EU/mL

mvc = AM — 0.065 EU/mL X_ 10-0 mL/Kg - 9.13 mL/mL
K 5.0 EU/Kg
MVD = Potency of Product _ _1.0 mL/mL = 1:7.7
MVC 0.13 mL/mL

INTRATHECAL DRUGS

Drug Administered on a Weight-per-Kilogram Basis

Product: Gentamicin Sulfate
Potency: 2.0 mg/mL

Maximum Dose/Kg ( M ): 0.11 mg/Kg
Lysate Sensitivity (A): 0.1 EU/mL

mvc =_AM  —_ 0.1 EU/mL X 0.11 mg/Kg = 9.055 mg/mL
K 0.2 EU/Kg
MVD = Potency of Product . _2.0 mg/mL — = 1:36.4
MVC 0.055 mg/mL

Drug Administered on a Volume-per-Kilogram Basis

Product: Lidocaine Hydrochloride Injection
Maximum Dose/Kg ( M ): 0.057 mL/Kg
Lysate Sensitivity (A): 0.1 EU/mL

mvc = -AM_ = 0.1 EU/ml X_ 0.057 mb/Ke = 0.0285 mL/mL
K 0.2 EU/Kge
mvp = Potency of Product = 1.0 mL/mL = 1:35.0

MVC 0.0285 mL/mL


II. LEGAL EFFECT OF THE GUIDELINE

This guideline is issued under section 10.90(b) (21 CFR 10.90(b)) of
FDA's administrative regulations, which provides for use of guidelines
to outline procedures or standards of general applicability that are
acceptable to FDA for a subject matter within its statutory authority.
Although guidelines are not legal requirements, a person who follows an
agency guideline may be assured that the Procedures or standards will be
acceptable to FDA. The following guideline has been developed to inform
manufacturers of human drugs (including biologicals), animal drugs, and
medical devices of procedures FDA considers necessary to validate the use
of LAL as an end-product endotoxin test. A manufacturer who adheres to
the guideline would be considered in compliance with relevant provisions
of the applicable FDA current good manufacturing practice regulations
(CGMP) for drugs and devices and other applicable requirements. As
Provided in 21 CFR 10.90(b), persons who use methods and techniques not
Provided in the guideline should be able to adequately assure, through
validation, that the method or technique they use is adequate to detect
the endotoxin limit for the product.


VI. APPENDICES

~ 16 -


Etidocaine HCI

Etidocaine HCI and Epinephrine
Etidronate Disodium

Etomidate Injection

Etoposide Injection

Evans Blue Injection

Factor IX
Famotidine
Fat Emulsion

Fentanyl Citrate
Fentanyl Citrate and Droperidol

Ferrous Citrate Fe59 Injection
Fibrinogen

Fibrinogen, Dried
Fibrinolysin and Desoxyribonuclease
Flurodopa F 18 Injection
Floxuridine
Fluorodeoxyglucose F18
Fluorescein Sodium Injection
Fluorouracil Injection
Fluphenazine HCl
Flupenthixol Decanoate

Folate Sodium

Folic Acid Injection

Fructose

Fructose and Sodium Chloride
Furosemide Injection

Gallamine Triethiodide
Gallium Citrate Ga67 Injection
Gentamicin Sulfate
*Gentamicin Sulfate

Globulins (Humans)

Glucagon for Injection

5.50 mg
5.50 mg
7.50 mg
0.60 mg
2.57 mg
0.36 mg

-F-

50.00 units
0.30 mg
(10%) 3.20 mL
(20%) 1.60 mL
0.10 mg
0.004 mg
Fentanyl
7.00 mL
30.00 mg
30.00 mg
1.00 units
7.00 mL
0.60 mg
7.00 mL
10.70 mg
15.00 mg
0.03 mg
0.57 mg
0.01 mg
0.014 mg
10.00 mL
10.00 mL
1.40 mg

-G-

1.00 mg
7.00 mL
3.00 mg
0.11 mg
1.00 mL
0.04 units

0.90
0.08
0.67
8.35
1.95 #
14.00 +

0.10
16.67
1.56
3.13
50.00
1250.00

25.00 +
0.17
0.17
5.00

25.00 +
8.33 #

25.00
0.47
0.33 #

166.67
8.77

500.00

357.10 +
0.50 +
0.50 +
3.60 +

5.00 +
25.00 +

1.70 +
45.46

5.00
125.00


Glycine Irrigation

Glycopyrrolate

Gold Au198 Injection

Gold Sodium Thiomalate Injection
Gonadorelin HCI

Haloperidol, Decanoate and Lactate
Hemin for Injection
Heparin Sodium and Calcium
Heparin Sodium Injection
Heparin Lock Flush Solution
Heparin and Sodium Chloride
Hetacillin Potassium
Hetastarch
Hexafluorenium Bromide Injection
Histamine Phosphate
Hyaluronate Sodium
Hyaluronidase Injection

and for Injection
Hydralazine HCI Injection
Hydrocortisone Suspension
Hydrocortisone Acetate
Hydrocortisone Sodium Phosphate
Hydrocortisone Sodium succinate
Hydromorphone HCL
Hydroxocobalamin
Hydroxyprogesterone Caproate
Hydroxystilbamidine Isethionate
Hydroxyzine HCI Injection
Hyocyamine Sulfate
Hyocyamine Sulfate and Scopolamine

Idarubicin HCI Injection
Ifosfamide

Imipenem

Imipenem and Cilastatin
Imipramine HCI Injection

--.-- mL
0.009 mg
7.00 mL
1.00 mg
1.40 meg

-H-

0.07 mg
4.00 mg
143.00 units
143.00 units
10.00 mL
10.00 mL
--.-- mg
20.00 mL
0.60 mg
0.04 mg

0.071 mg

2.14 units
3.50 mg
4.00 mg
1.07 mg
4.00 mg
4.00 mg
0.057 mg
14.30 mcg
14.30 mg
4.50 mg
1.40 mg
0.007 mg
0.007 mg

“Ie

0.31 mg
30.86 mg
-.-- mg
7.14 mg
1.00 mg

43

0.50 +
555.50 +
25.00 +
5.00
3.60

71.40 +
1.25
0.03
0.03
0.50
0.50
0.30 +
0.25
8.35

125.00 +

70.42

2.30 +
1.45 +
1.25 +
4.67
1.25 +
1.25 +
88.00 +
0.40 +
0.35
1.10 +
3.60 +
714.30 +
714.29

16.13 #
0.16 #+
0.23 +
0.70
5.00 +


EXAMPLE:
RSE Standard Curve

Straight Line Equation (Y) = 3.03 + (-0.18] * X)
RSE Standard Range = 1037 - 2235 seconds (17.3-37.3 minutes)

CSE Standard Curve

Endotoxin Concentration(ng/mL)

Vial O.1 0.05 0.025 0.0125 0.006 0.003
1 1018.8 1114 1218.6 1402.7 1548.7 1740.7
2 990.7 1090.6 1249.8 1406.4 1586.0 1780.0
3 998.2 1116.8 1227.8 1411.0 1554.1 1800.9
4 1003.4 1086.1 1198.5 1415.6 1593.9 1781.0

Note: Each T in the above table is expressed in seconds and
represents the mean of at least duplicate determinations.

Mean T (sec.) 1002.8* 1101.9 1223.7 1408.9 1570.7 1775.7
Log mean T 3.001 3.042 3.088 3.149 3.196 3.249

Calculations:

Solving for EU/mL equivalent by substituting onset times generated with
CSE (ng/mL) into the above RSE standard line equation, X = ( Y —
3.03)/-0.181 where ¥ = log mean onset time and X = log EU/ml equivalent.

EU/mL Equivalent

CSE Endo. Conc. Log Mean (RSE Std. Line) EU/ng
(ng/mL) T Log Antilog
O.1* 3.001 0.16 1.45 14.5
0.05 3.042 -0.066 0.859 17.2
0.025 3.088 -0.32 0.479 19.2
0.0125 3.149 -0.657 0.22 17.6
0.006 3.196 -0.917 0.121 20.2
0.003 3.249 -1.210 0.062 20.6

Mean EU/ng = 19.0 (SD = 1.52)
* Outside the RSE standard range - not used in calculation of mean.

The values for the y-intercept and slope of the four CSE curves used for
the EU/ng determination may be stored for use in routine testing
(archived standard curve) instead of running a series of standards each
day. Using the EU/ng conversion factor, CSE standards within the range
of the RSE curve can be made up in endotoxin units. Standards outside
this range require the use of RSE and a new RSE standard curve. If CSE
standards outside the RSE standard range are required the EU/ng
conversion factor must be determined for the new range as described
above.

-~21-


CONTENTS

INTRODUCTION

I. Background

Il. Legal Effect... ccc eee e ccc e ccc eect eee eee ete eee ete e ete enees 3

III. Regulatory RequirementsS.......... cece cece eee eee ete eee eeee 4

Iv. Human and Animal Drugs and Biological Products.............0e00 6
A. Validation of the LAL Test.......... 0.2 cece cece ee eee eens 6

VI.

B. Testing of Devices by the LAL ceSt.......... ccc cece ee eee eee 14
9 2) of 5 00 6 ok 1 16
A. Initial Quality Control....... eee eee eee cece ee eens 17

B. USP XXI/NF XIV Bacterial Endotoxins Test....
C. Determination of the Relationship Between the Control
Standard Endotoxin and the Reference Standard Endotoxin..... 19

D. Maximum Valid Dilution Calculation... ele eee cece 22



Propranolol HCI Injection
Protamine Sulfate Injection
Protein Hydrolysate Injection
Prothrombin Complex
Protirelin

Pyridostigmine Bromide
Pyridoxine HCI

Quinidine Sulfate
Quinidine Gluconate

Ranitidine HCI

Reserpine

Riboflavin

Rifampin for Injection
Ringer’s Injection

Ringer’s Irrigation

Ringer’s in Dextrose
Ringer’s - Lactated Injection
Ringer’s - Lactated in Dextrose
Ritodrine HCl
Rolitetracycline for Injection
Rolitetracycline Nitrate

Saralasin Acetate

Secretin

Scopolamine Butylbromide
Scopolamine HBr
Secobarbital Sodium Injection
Selenious Acid (Selenium)
Selenomethionine Se75 Injection
Sincalide

Sisomicin Sulfate

Sodium Acetate

Sodium Ascorbate

0.09 mg
0.71 mg
10.00 mL
50.00 units
7.00 meg
0.29 mg

14.29 mg

-Q-

8.60 mg
8.60 mg

-R-

0.07 mg
0.70 mg
-.-- mg
10.00 mL
10.00 mL
10.00 mL
10.00 mL
10.00 mL
5.00 mg
5.00 mg

-S-

0.26 mg
1.00 unit
0.29 mg
0.009 mg
5.50 mg
1.43 ug
7.00 mL
0.02 mcg
10.00 mg
1.29 mEq
3.57 mg

50

55.60 +
7.04 +
0.50 +
0.10
0.70

17.00 +
0.40 +

0.60 +
0.60

7.00
71.50 +
7.10 +
0.20 +
0.50 +
0.50 +
0.50
0.50 +
0.50
0.50 +
1.00 +
1.00

19.20
5.00
17.24
555.60 +

0.90 +
3.50 +
25.00 +
250.00
0.50 +
3.90 +
1.40


-O-

Opium Alkaloids HCI 0.28 mg
Orphenadrine Citrate Injection 0.86 mg
Ouabain 0.007 mg
Oxacillin Sodium 25.00 mg
Oxymorphone HCI 0.021 mg
Oxytetracycline 12.50 mg
Oxytocin 0.14 units
-P-

Pancuronium Bromide 0.10 mg
Papaverine HCI 1.70 mg
Paraldehyde 0.15 mL
Parathyroid Hormone 0.57 units
Penicillin G Benzathine Suspension 50,000.00 units
Penicillin G Potassium 50,000.00 units
Penicillin G Procaine

and Suspension 50,000.00 units

Penicillin G Procaine, Dihydrostreptomycin
Sulfate, Chlorpheniramine Maleate and

Dexamethasone Suspension --.-- units
Penicillin G Procaine, Dihydrostreptomycin

Sulfate, Prednisolone Suspension --.-- units
Penicillin G Sodium 50,000.00 units
Pentagastrin 0.006 mg
Pentamidine Isethionate 4.00 mg
Pentobarbital Sodium Injection 6.00 mg
Pentazocine Lactate Injection 0.86 mg
Perphenazine 0.14 mg
Phenobarbital Sodium Injection 20.00 mg
Phenolsulfonphthalein 0.09 mg
Phentolamine Mesylate 0.86 mg
Pentylenetetrazol 7.14 mg
Phenylephrine HCI 0.20 mg
Phenytoin Sodium Injection 20.00 mg
Physostigmine Salicylate 0.06 mg
Phytonadione 0.36 mg
Piperacillin Sodium 75.00 mg
Piperocaine HCI 4.30 mg

48

17.86
5.80 +
714.29
0.20 +
238.10 +
0.40 +
35.70

50.00

2.90 +
33.33

8.80
0.01/100 +
0.01/100 +

0.01/100 +

0.01/100 +

0.01/100 +
0.01/100 +
833.00
1.25
0.83 +
5.80 +
35.70 +
0.30 +
55.60
5.80 +
0.70
25.00 +
0.30 +
83.40 +
14.00 +
0.07 +
1.16


assay procedure. On pooled samples, if any endotoxin is detected in
the first repeat, proceed to second repeat. The second repeat
consists of an additional 10 units tested individually. None of the
10 units tested in the second repeat may contain endotoxin in excess
of the limit concentration for the drug product.

The following should be considered the endotoxin limit for all
parenteral drugs to meet if the LAL test is to be used as an
end-product endotoxin test:

1. K/M: For any parenteral drug except those administered
intrathecally, the endotoxin limit for endotoxin is
defined as K/M, which equals the amount of endotoxin
(EU) allowed per ng or mL of product. K is equal to 5.0
EU/Kg. (SEE appendix D for definition of M).

For parenteral drugs that have an intrathecal route of
administration, K is equal to 0.2 EU/Kg.

Drugs exempted from the above endotoxin limits are:

1. Compendial drugs for which other endotoxin limits have been
established.

2. Non-compendial drugs covered by new drug applications,
antibiotic drug applications, new animal drug applications, and
biological product licenses where different limits have been
approved by the agency.

3. Investigational drugs or biologicals for which an IND or INAD
exemption has been filed and approved.

4. Drugs or biologicals which cannot be tested by the LAL method.

A batch which fails a validated LAL release test should not be retested
by the rabbit test and released if it passes. Due to the high
variability and lack of reproducibility of the rabbit test as an
endotoxin assay procedure, we do not consider it an appropriate retest
procedure for LAL failures.


The sensitivity of the LAL technique used should be determined
by the procedures and criteria in Appendix A. Routine
performance of the LAL test should include standards (run in
duplicate) and a negative control. An endotoxin standard series
is useful for checking lysate sensitivity and the competence of
the technician, and for identifying other problems such as the
contamination of glassware,

The stability of the endotoxin standards and appropriate storage
conditions should also be considered; dilute endotoxin solutions
are not as stable as more concentrated solutions under certain
conditions.

INHIBITION AND ENHANCEMENT TESTING

Lack of product inhibition or enhancement of the LAL test should
be shown for each type of device before use of the LAL test.
Possible inhibition of different chemical components of similar
devices should be considered. A manufacturer may logically
divide its device products into groups of products according to
common chemical formulation; and may then qualify only a
representative product from each such group. Ideally, the
product chosen from each group would be the one with the largest
surface area contacting body or fluid for administration to a
patient.

At least three production lots of each product type should be
tested for inhibition. In general, use of the sampling
technique selected should result in a random sampling of a
finished production lot. CDRH recommends testing 2 devices for
lot sizes under 30, 3 devices for Jot sizes 30-100, and 3
percent of lots above size 100, up to a maximum of 10 devices
per lot.

The process of preparing an eluate/extract for pyrogen or
inhibition/enhancement testing may vary for each device. Some
medical devices can be flushed, some may have to be immersed in
the non-pyrogenic rinse solution, while others may be tested by
disassembling or by cutting the device into pieces prior to
extraction by immersion. In general, for devices being flushed,
the non-pyrogenic rinse solution should be held in the fluid
pathway for one hour at room temperature (above 18° C);
effluents should be combined. If a device is to undergo
extraction, a minimum extraction time should be 15 minutes at
37° C, one hour at room temperature (above 18° C) or other
demonstrated equivalent conditions.

Guidelines for rinse volumes include the following:

a. Each of the 10 test units should be rinsed with 40 mL of
non-pyrogenic water.


GUIDELINE ON
VALIDATION OF THE LIMULUS AMEBOCYTE LYSATE TEST
AS AN END~PRODUCT ENDOTOXIN TEST FOR HUMAN
AND ANIMAL PARENTERAL DRUGS, BIOLOGICAL PRODUCTS, AND
MEDICAL DEVICES

December 1987

Prepared by: Center for Drug Evaluation and Research
Center for Biologic Evaluation and Research
Center for Devices and Radiological Health
Center for Veterinary Medicine

Maintained by: Division of Manufacturing and Product Quality (HFN-320)
Office of Compliance
Center for Drug Evaluation and Research
Food and Drug Administration
5600 Fishers Lane
Rockville, MD 20857


Dopamine HCI

Dopamine HCI in Dextrose
Doxapram HCI Injection
Doxorubicin HC! for Injection
Doxycycline Hyclate for Injection
Dromostanolone Propionate
Droperidol

Dyphylline Injection

Edetate Calcium Disodium

Edetate Disodium

Edrophonium Chloride Injection

Electrolyte Solutions- LVP

Multiple Electrolytes Type 1 & 2

Multiple Electrolytes and Invert Sugar
Type 1,2, and 3

Multiple Electrolytes and Dextrose
Type 1,2,3,and 4

Emetine HCI

Enalaprilat

Ephedrine Sulfate Injection

Epinephrine Injection

Epinephrine Suspension

Ergocalciferol (D2)

Ergoloid Mesylates

Ergonovine Maleate

Ergotamine Tartrate

Erythromycin Gluceptate/Lactobionate

Esmolol

Estradiol (aqueous)

Estrogens (Combined) Aqueous

Estrogens Conjugated

Estrogenic Substances or Estrogens
Estrone Aqueous Suspension
Ethacrynate Sodium

Ethamivan Injection
Ethylnorepinephrine HCI Injection

0.30 mg
0.30 mg
1.50 mg
1.93 mg
4.40 mg
1.40 mg
0.14 mg
7.10 mg

-E-

50.00 mg
25.00 mg
0.60 mg
10.00 mL
--.-- mL

--.-- mL

--.-- mL
0.93 mg
0.018 mg
3.00 mg
0.014 mg
0.025 mg
142.80 units
0.004 mg
6.00 meg
0.014 mg
5.00 mg
0.50 mg
0.02 mg
0.026 mg
Estrone
0.36 mg
0.057 mg
0.057 mg
1.00 mg
1.40 mg
0.029 mg

41

16.67 +
16.67 +
3.30 +
2.20 #+
1.14 +
3.57

35.70
0.70 +

0.10 +
0.20
8.33 +
0.50
0.50

0.50

0.50
5.40 +
280.00
1.70 +
357.00 +
200.00
0.035
1250.00
0.80
357.00 +
1.00 +
10.00
250.00 +
192.31

14.00
88.00
88.00 +
5.00 +
3.60
172.40 +


APPENDIX C

DETERMINATION OF THE RELATIONSHIP BETWEEN THE
CONTROL STANDARD ENDOTOXIN (CSE) AND THE REFERENCE
STANDARD ENDOTOXIN (RSE)

If a manufacturer chooses to use an endotoxin preparation (CSE) other than
the United States Pharmacopeia Reference Standard Endotoxin (RSE), the CSE
will have to be standardized against the RSE. If the CSE is not a
commercial preparation which has been adequately characterized, it should be
studied and fully characterized as to uniformity, stability of the
preparation, etc. The relationship of the CSE to the RSE should be
determined prior to use of a new lot, sensitivity, or manufacturer of the
LAL or a new lot source or manufacturer of the CSE.

A. GEL-CLOT TECHNIQUE

The following is an example of a procedure to determine the
relationship of the CSE to the RSE:

At least 4 samples (vials) for the lot of CSE should be assayed.
State in ng/mL the endpoint for the CSE and in EU/mL of the RSE.
The values obtained should be the geometric mean of the endpoints
using a minimum of 4 replicates.

Example: LAL end, vints for the RSE and CSE are as follows:

RSE = 0.3 EU/mL
CSE = 0.018 ng/mL

The EUs per ng of CSE are calculated as follows:

RSE = 0.3 EU/mL  — 16.7 EU/ng
CSE 0.018 ng/mL

This indicates that 0.018 ng of the CSE is equal to 0.3 EU of
the RSE. Thus, the CSE contains 16.7 EU/ng.

B. CHROMOGENIC AND ENDPOINT-TURBIDIMETRIC TECHNIQUES

At least 4 samples (vials) for the lot of CSE should be assayed.
In addition to a water blank, assay dilutions of RSE which fall in
the linear range and dilutions of the CSE. Linear regression
analysis is performed on the absorbance values of the RSE standards
(y-axis) versus their respective endotoxin concentrations
(x-axis). Calculate the EU/ng of the CSE by inserting the average
CSE 0.D. readings for each concentration which falls in the RSE
standard range into the RSE straight line equation. The resulting
CSE values (in EU) are then divided by their corresponding
concentrations (in ng/mL). These values are then averaged to
obtain the potency of the CSE lot.

- 19 -


S

ONAL |

4
‘
me

GUIDELINE ON
VALIDATION OF THE LIMULUS AMEBOCYTE LYSATE TEST .
AS AN END-PRODUCT ENDOTOXIN TEST FOR HUMAN
AND ANIMAL PARENTERAL DRUGS, BIOLOGICAL PRODUCTS, AND
MEDICAL DEVICES

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES,
PUBLIC HEALTH SERVICE
FOOD AND DRUG ADMINISTRATION


Triethylenethiophosphoramide
Triethylperazine Maleate
Trifluoperazine HCI Injection
Triflupromazine HCI Injection
Trimethaphan Camsylate
Trimethobenzamide HCI
Tromethamine

Tubocurarine Chloride

Urea
Urofollitropine
Urokinase

Vancomycin HCI

Vasopressin

Vecuronium Bromide
Verapamil Hydrochloride
Vidarabine for Injection
Vinblastine Sulfate for Injection
Vincristine Sulfate for Injection
Viomycin Sulfate

Vitamin A

Warfarin Sodium for Injection
Water for Injection and Sterile WFI
Bacteriostatic WFI

Sterile Water for Inhalation

Sterile Water for Irrigation

Xenon Xe133 Injection

*Ytterbium Yb169 Pentetate Injection
Zinc Chloride Injection

Zinc Sulfate Injection

0.80 mg
0.43 mg
0.029 mg
0.86 mg
5.00 mg
2.80 mg
154.00 mg
0.50 mg

-U-

1500.00 mg
1.06 units
4,400.00 TU

-V-

15.00 mg
0.14 units
0.10 mg
0.30 mg

10.00 mg
0.50 mg
0.05 mg

14.30 mg

714.30 TU

-XYZ-

2.00 mL
2.50 mL
0.20 mg Zn
0.20 mg Zn

53

6.25
11.63
172.00 +
5.80 +
1.00 +
1.80 +
0.03 +
10.00 +

0.003 +
4.70
0.002

0.33 +
35.70
50.00
16.70 +

0.50 +
10.00 #+

100.00 #

0.35

0.007

24.00 +
0.25 +
0.50 +
0.50 +
0.25 +

87.50 +
5.60 +
25.00 +
25.00 +


Bleomycin Sulfate

Bretylium Tosylate Injection

Bretylium Tosylate in Dextrose

Brompheniramine Maleate Injection

Bumetanide

Bupivacaine Hydrochloride Injection

Bupivacaine Hydrochloride and
Epinephrine Injection

Bupivacaine HCI and Dextrose

Buprenorphine HCI

Butorphanol Tartrate

Caffeine Citrated
Caffeine and Sodium Benzoate
Calcitonin - Human
Calcitonin - Salmon
Calcitriol
Calcium Ascorbate
Calcium Chloride
Calcium Disodium Edetate
Calcium Gluceptate Injection
Calcium Gluconate
Calcium Glycerophosphate
and Calcium lactate
Calcium Levulinate
Capreomycin Sulfate
Carbazochrome Salicylate
Carbenicillin Disodium
Carboplatin
Carboprost Tromethamine
Carmustine for Injection
Cefamandole Nafate and Sodium
Cefazolin Sodium
Cefmetazole Sodium
Cefonicid Sodium
Cefoperazone Sodium
Ceforanide
Cefotaxime Sodium
Cefotetan Disodium

0.50 unit
25.00 mg
25.00 mg

0.14 mg

0.01 mg

2.50 mg

3.20 mg
0.11 mg
0.004 mg
0.057 mg

-C-

20.00 mg
7.14 mg
0.007 mg
4.00 TU
0.05 meg

14.30 mg

25.00 mg

35.00 mg

15.70 mg

28.60 mg

1.43 mg
0.14 mL
14,30 mg
0.14 mg
100.00 mg
9.26 mg
0.007 mcg
5.14 mg
33.30 mg
33.30 mg
-.-- mg
14.30 mg
28.57 mg
20.00 mg
28.50 mg
28.60 mg

37

10.00 #+
0.20
0.20

35.71

500.00 +
2.50 +

1.60 +

1.80 +
1250.00

88.00 +

0.25
0.70 +
714.30
1.25
100.00
0.35
0.20 +
0.143
0.32 +
0.17 +

3.50
35.70 +
0.35 +

34.96
0.05 +
0.54 #
714.30 +
1.00 #
0.15 +
0.15 +
0.20 +
0.35 +
0.20 +
0.25 +
0.20 +

0.17


Menadione

N. Meningococcal Polysaccharide
Pur. Bulk, Group A

N.Meningococcal Polysaccharide
Pur. Bulk, Group C

Meningococcal Polysaccharide Vaccine
Group A

Meningococcal Polysaccharide Vaccine
Group C

Meningococcal Polysaccharide Vaccine
Group A and C

Menotropin

Meperidine HCI Injection

Mephentermine Sulfate

Mepivacaine HCI

Mepivacaine HCI and Levonordefrin

Meprobamate Injection

Meprylcaine HCI and Epinephrine

Mercaptomerin Sodium

Mersalyl with theophylline

Merethoxylline Procaine

Mesoridazine Besylate Injection

Metaraminol Bitartrate

Methadone HCI

Methandroil

Methapyrilene HCl

Methicillin Sodium

Methiodal Sodium Injection

Methocarbamol Injection

Methohexital Sodium

Methotrexate Sodium Injection

Methotrimerprazine

Methoxamine HCI

Methyldopate HCl

Methylene Blue Injection

Methylergonovine Maleate

Methylprednisolone Acetate Sus

Methylprednisolone Sodium Succinate
for Injection

Metoclopramide

Metocurine Iodide

0.09 mg
0.25 ug
0.25 ug
0.025 ug
0.025 ug

0.05 ug
2.00 units
2.14 mg
0.64 mg
6.60 mg
6.60 mg
1.00 mg
6.60 mg
3.57 mg
2.90 mg
2.90 mg
0.71 mg
1.43 mg
0.57 mg
1.43 mg
0.60 mg
50.00 mg
1300.00 mg
28.60 mg
10.00 mg
-.-- mg
0.28 mg
0.25 mg
10.00 mg
2.00 mL
2.90 meg
0.80 mg

30.00 mg

2.00 mg
0.40 mg

46

58.30 +

20.00

20.00

200.00

200.00

100.00
2.50 +
2.40 +
7.80 +
0.80 +
0.80 +
5.00
0.80 +
1.40
1.72
1.72
7.00 +
3.50 +
8.80 +
3.50
8.33
0.10 +
0.004
0.20 +
0.50 +
2.00 +

17.90 +
20.00 +
0.50 +
2.50 +
1.70 +
6.25

0.17 +
2.50 +
12.50 +


APPENDIX D

MAXIMUM VALID DILUTION

To determine how much the product can be diluted and still be able to
detect the limit endotoxin concentration, the following two methods will
determine the Maximum Valid Dilution:

METHOD I

This method is used when there is an official USP limit or when the
limits listed in Appendix E are used.

For

Mvp = Endotoxin Limit X Potency of Product

drugs administered on a weight-per-kilogram basis, the

potency is expressed as mg or units/mL and for drugs
administered on a volume-per-kilogram basis, the potency is
equal to 1.0 mL/mL.

METHOD IT

This method is used when there is no official USP limit and the
limits listed in Appendix E are not used.

Step l.

Where:

Minimum Valid Concentration (MVC)

MVC = AM
K

GEL CLOT: Labeled sensitivity-EU/mL.
CHROMOGENIC, TURBIDIMETRIC and KINETIC-TURBIDIMETRIC:
The lowest point used in the standard curve.

Rabbit Dose or Maximum Human Dose/Kg of body weight that
would be administered in a single one hour period, whichever
is larger. For radiopharmaceuticals, M equals the rabbit
dose or maximum human dose/Kg at the product expiration date
or time. Use 70 Kg as the weight of the average human when
calculating the maximum human dose per Kg. Also, if the
pediatric dose/Kg is higher than the adult dose then it
shall be the dose used in the formula.

5.0 EU/Kg for parenteral drugs execpt those administered
intrathecally; 0.2 EU/Kg for intrathecal drugs

~ 22 -


inhibition or enhancement, the drug product can be diluted,
mot to exceed the MVD, and the test repeated.

An alternate procedure may be performed whereby the RSE/CSE
standard curve is prepared in drug product or product
dilution instead of water. The drug product cannot have a
background endotoxin concentration of more than 10%
(estimated by extrapolation of the regression line) of the
lambda concentration (lambda equals the lowest concentration
used to generate the standard curve). The standard curve
must meet the test for linearity, i.e. r equal to or less
than -0.980, and in addition the slope of the regression
must be less than -0.1 and greater than -1.0. If the
standard curve does not meet these criteria, the drug
product cannot be tested by the alternate procedure.

In those instances when the drug is manufactured in various
concentrations of active ingredient while the other components of the
formulation remain constant, only the highest and lowest concentration
need be tested. If there is a significant difference, i.e. greater
than twofold, between the inhibition endpoints or if the drug
concentration, per mL, in the test solutions is different, then each
remaining concentrations should be tested. If the drug product shows
inhibition or enhancement at the MVD, when tested by the procedures in
the above sections, and is amenable to rabbit testing, then the rabbit
test will still be the appropriate test for that drug. If the
inhibiting or enhancing substances can be neutralized without
affecting the sensitivity of the test or if the LAL test is more
sensitive than the rabbit pyrogen test the LAL test can be used. For
those drugs not amenable to rabbit pyrogen testing, the manufacturer
should determine the smallest quantity of endotoxin that can be
detected. This data should be submitted to the appropriate FDA Office
for review.

The inhibition/enhancement tests must be repeated on one unit of the
product if the lysate manufacturer is changed. If the lysate
technique is changed, the inhibition and enhancement tests must be
repeated using three batches. When the manufacturing process, the
product formulation, the source of a particular ingredient of the drug
formulation, or lysate lot is changed, the positive product control
can be used to reverify the validity of the LAL test for the product.
Firms that are obtaining an ingredient from a new manufacturer are
encourged to include as part of their vendor qualification the rabbit
Pyrogen test to determine that the ingredient does not contain
non~endotoxin pyrogens.

Routine Testing of Drugs by the LAL Test.

End-product testing is to be based on data from the
inhibition/enhancement testing as outlined in Section A(2). Samples,
Standards, positive product controls and negative controls should be
tested at least in duplicate.


(*) - Intrathecal Injections

(+) - USP Limit
NOTE: The limit formula for radiopharmaceuticals is 175/V except for
intrathecally administered products 14/V for intrathecal products. V
equals the maximum recommended dose (listed in the dose column), in
mL, at the expiration date or time.

(® - Drug Administered on a per Square Meter of Body Surface
Limit calculated according to the following formula:
5 EU/Kg / ((dose * 1.80 sq. m.)/70 Kg)

References:

Facts and Comparisons, Editors E. Kastrup and J. Boyd,
Facts and Comparisons, Inc.

United States Pharmacopeia Dispensing Information
1990, United States Pharmacopeia Convention, Inc.

54


V. MEDICAL DEVICES

General Requirements

The CDRH has reviewed the results of the "HIMA Collaborative Study for
the Pyrogenicity Evaluation of a Reference Endotoxin by the USP Rabbit
Test." This study recommends 0.1 ng/mL (10 mL/kg) of Es co 055:B5
endotoxin from Difco Laboratories as the level of endotoxin which should
be detectable in the LAL test when used for end-product testing of
medical devices. This sensitivity (0.1 ng/mL given 10 mL/kg) is
sufficient for LAL testing and for retest of devices in rabbits.
According to recent collaborative studies in the rabbit pyrogen and LAL
tests, one nanogram of E. coli 055:B5 endotoxin is similar in potency to
5 EU of the USP Endotoxin Reference Standard. The endotoxin limit for
medical devices has been converted to EU and is now 0.5 EU/mL using the
rinse volume recommended in Section 2 below. Liquid devices should be
more appropriately validated and tested according to the requirements for
drugs by taking the maximum human dose per kilogram of body weight per
hour into consideration (See Section IV,B).

Manufacturers may retest LAL test failures with the LAL test or a USP
rabbit pyrogen test. If the endotoxin level in a device eluate has been
quantitated by LAL at 0.5 EU/mL endotoxin or greater, then retest in
rabbits is not appropriate. Medical devices that contact cerebrospinal
fluid should have less than 0.06 EU/mL of endotoxin. These values
correspond to those set by the CDER for intrathecal drugs.

Manufacturers shall use an LAL reagent licensed by OBRR in all
validation, in-process, and end-product LAL tests.

A. Validation of the LAL Test

1. Sensitivity: Data demonstrating the sensitivity and
reproducibility of the LAL test.

2. Inhibition/Enhancement Testing: Each product line of devices
utilizing different materials or metnods of manufacture should
be checked for inhibition or enhancement of the LAL test.

Further explanation of the above points is given as follows:
1. SENSITIVITY

A manufacturer must be able to demonstrate a sensitivity of at
least 0.5 EU/mL. The level of endotoxin selected as the
pass/fail point for evaluating pyrogenicity of products using
the LAL test must be equivalent to or below this level.
Manufacturers may use another endotoxin if a _ reproducible
correlation between it and the USP Reference Endotoxin Standard
has been demonstrated in their laboratory (see appendix C).