BACTERIAL ENDOTOXIN TEST (BET)/ LIMULUS AMEBOCYTE LYSATE (LAL)

1. Background on Endotoxins

 Endotoxin

 A heat-stable toxin associated with the outer membranes of certain gram-negative

bacteria. Endotoxins are not secreted but are released only when the cells are
disrupted; they are less potent and less specific than the Exotoxins.

 In large quantities they produce hemorrhagic shock and severe diarrhea; smaller
amounts cause fever, altered resistance to bacterial infection, leukopenia followed by
leukocytosis, and numerous other biologic effects.

2. History of Bacterial endotoxin Test

 Prior to WWII, Rabbit pyrogen test introduced

 1956 Dr. Bang discovered that horseshoe crab blood forms clots when
bacteria (Vibrio sp.) are present
 1968 Dr. Bang and Dr. Levin created Limulus amoebocyte lysate, or LAL,
and a new method to test for gram-negative bacteria
 1971 LAL assay is developed
 1977 FDA replaces Rabbit test with LAL test
 1987 The FDA establishes guidelines for LAL testing of pharmaceuticals
and medical devices
 2012 FDA established the guide lines for the endotoxin
(https://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/
Guidances/UCM310098.pdf)

 2016 FDA need endotoxin test for medical devices

3. Bacterial endotoxin test methods

The Limulus Amebocyte Lysate (LAL) test is an in vitro assay used for detection
of pyrogenic substances (endotoxin) in sterile parenteral drugs, in-process manufacturing
samples, cleaning validation rinse samples, and medical devices. Limulus amebocyte lysate is
an aqueous extract of blood cells (amebocytes) from the horseshoe crab, Limulus
polyphemus. The LAL test can be performed by any of the following methods:

 Gel-Clot method
 Kinetic Turbidimetric

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  Kinetic Chromogenic

Selection of the most suitable LAL test method is based on the assay results obtained during
the assay development and validation.

Gel-Clot LAL Test

The Gel-Clot LAL test is performed by adding the lysate and the test specimen in a pyrogen-
free reaction tube. The reaction solution is placed immediately in a dry block incubator or
water bath at 37 ± 1°C for 60 ± 2 minutes. After 60 minutes the tube is removed from the
incubator and inverted. A positive test is a firm gel (clot) that remains intact in the bottom of
the reaction tube after inversion of 180°. The concentration of endotoxin in the tube is greater
than or equal to the sensitivity of the lysate. Lack of a gel indicates a negative test. The Gel-
Clot LAL test can detect as little as 0.015 EU/per ml.

Kinetic Turbidimetric LAL Test

In the Kinetic Turbidimetric LAL method, either the rate of increase in turbidity or the time
taken to reach a particular level of turbidity (the onset time) is determined. The assay requires
a 96 well plate reader to incubate multiple samples at a controlled temperature (37°C) and
takes optical density readings at regular intervals. Standard curves are prepared by plotting
the log onset time against the log concentration of standard endotoxin and are used to
calculate endotoxin concentrations in specimens. The sensitivity of this test is 0.01 EU/mL.

Chromogenic LAL Test

In the Chromogenic LAL test, the LAL reagent is mixed with test sample in a microplate and
incubated in a reader at 37 ± 1°C. Absorbance measurements are collected with time after
addition of Chromo-LAL and analyzed by the K-QCL software. The time (onset time) taken
for a sample to reach a specified absorbance (onset OD) is calculated; and a standard curve,
showing the linear correlation between the log onset time and the log concentration of
standard endotoxin, is generated. The sensitivity of this test is 0.005 EU/mL.

4. Official Endotoxin Testing Sources

 USP <85> Bacterial Endotoxin Test

 USP <161>Bacterial Endotoxin Test for Transfusion and infusion assemblies and similar
medical devices(Proceed as directed under <85> as additional guidance)
 ANSI/AAMI ST72- Bacterial Endotoxin Test
 EP 2.6.14 Endotoxin Test
 FDA Guidance for Industry Pyrogen and Endotoxin Testing: Questions and Answers
(https://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/
Guidances/UCM310098.pdf)

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 5. Gap Analysis between USP<85> and USP <161>

Specification USP <85> USP <161>

Transfusion and infusion
Drug Injection/Biological products
assemblies and medical devices
Test category
(Injectable drugs and powder
(sterilized products, medical
samples)
implants)
Does not recommend how many lots
Lot/Sample Select not less than 3 and not more
or samples should be tested for each
size selection than 10 devices
production type
-0.5 EU/ml or 20 EU/device for
products that directly or indirectly
contact the cardiovascular system
Endotoxin Does not recommend specific limits
limits for medical devices
-0.06EU/ml or 2.15 EU/device for
devices in contact with
cerebrospinal fluid
Test methodologies, routine monitoring and alternatives to batch testing by AAMI ST72

6. Sample selection

Selection of Number of Samples

Lot Size Number of Samples
<30 2
30-100 3
>101 3% of lot, up to a maximum of 10