TECHNICAL TIP

BACTERIAL ENDOTOXIN TESTING

Pyrogens are substances that can induce a fever response in the Samples are defined in USP<85>, USP<161>, USP<1085> as:
body, and sources can be either microbial or non-microbial. The
• 3-10 samples
focus of this TechTip is the testing associated with a microbial
pyrogen, bacterial endotoxin. Additional alert and action levels may be established by the
manufacturer to monitor and trend, assuring control of the
Bacterial endotoxins are membrane bound lipopolysaccharides
manufacturing process.
in the cell wall of Gram-negative bacteria. The toxicity of these
lipopolysaccharides is attributed to the Lipid A component of the Bacterial endotoxins are heat stable chemicals and may exist after
lipopolysaccharide. the death or sterilization of the bacterial source. In living bacteria,
endotoxin can be continuously released by the cell. Given the size
The Bacterial Endotoxin Test is used to measure pyrogenic
of the endotoxin, filtration is generally not an effective method of
(fever-inducing) substances derived from Gram-negative
removal. Strong acids, strong bases or high temperatures may be
bacterial contamination. Within the context of medical device
required to reduce endotoxin contamination on a product. These
and pharmaceutical manufacturing, Gram-negative bacterial
methods, however, can have adverse effects on common medical
contamination is generally isolated from water sources. If certain
device component materials.
levels of these chemicals are present on or in the medical device
or pharmaceutical, they can lead to significant patient harm or The reagent used in the Bacterial Endotoxin Test, Tachypleus
death depending on the type of patient contact. It is critical that or Limulus Amebocyte Lysate (TAL/LAL), is isolated from the
devices with contact to the circulatory system, cerebrospinal hemolymph of either the Southeast Asian (Tachypleus) or Atlantic/
fluid, the eyes, implants, and other parts of the body that may American (Limulus) genus of horseshoe crab.
be susceptible to endotoxin be monitored regularly to ensure
Utilizing the clotting factor of the horseshoe crab, the Bacterial
patient safety.
Endotoxin Test utilizes the combination of a liquid or liquid
Details regarding endotoxin testing are found in Pharmacopeias extract of a product/device combined with the TAL/LAL reagent
(USP<85>, USP<161>, USP<1085>, EP 2.6.14, etc.). to detect or quantify bacterial endotoxin in the sample through
multiple methods.
Limits are described in ANSI/AAMI ST72, Bacterial Endotoxins
– Test Methods, Routine Monitoring, and Alternatives to Batch Extractions for endotoxin are typically performed on the patient-
Testing, for the following types of devices: contacting portions of the device (direct or indirect contact)
or those labeled as non-pyrogenic. Extractions are typically
Device Type Limit (Endotoxin Units/device) performed for either 15 minutes at 37°C or one hour with 37°C
Circulatory Contacting < 20 EU/device
water extracted at room temperature. Intraocular products
may require one hour extraction at 37°C with agitation due to
Cerebrospinal Fluid Contacting < 2.15 EU/device
endotoxin sensitivity in the eyes. Testing of process water does
Intraocular Devices < 0.2 EU/device
not require extraction. Some materials may inhibit or enhance
the result due to the presence of beta glucans. Beta glucan
Samples are described in ANSI/AAMI as:
interference may require special buffers to ensure accurate results.
• < 30 batches: 2 samples

• 30-100 batches: 3 samples

• ≥ 101 batches: 3% of batch up to a maximum of 10

samples

F O R M O R E I N F O R M AT I O N
STERIS Applied Sterilization Technologies
Web: www.steris-ast.com // Email: [email protected]
(EMEA-APAC) +44 (0) 8456 88 99 70
(Americas) 877.783.7479

TechTip #66 | Rev 1, 3/2023

 TECHNICAL TIP

BACTERIAL ENDOTOXIN TESTING

Product release testing for endotoxin may be tested either prior the sample and correlates to the amount of endotoxin in the
to or after sterilization. The risk in testing prior to sterilization sample (established using a standard curve of known endotoxin
comes from potential changes in Gram-negative bioburden concentrations). Time taken to reach a specific turbidity level is
levels between the time of testing and the time of sterilization also considered in the measurement.
that consequently could impact endotoxin levels. Risk may be
The Kinetic turbidimetric method is not suitable for samples with
increased in samples containing water or nutrients. It is important
color, turbidity, or high viscosity.
to document this risk assessment if testing endotoxin prior to
terminal sterilization to ensure patient safety. Kinetic Chromogenic

There are different methods of analysis that can be performed The chromogenic method of analysis uses a chemically
based on modifications that are made to the TAL/LAL reagent. induced color change as a measurement of endotoxin. Like the
The selected method of analysis for endotoxin testing must be turbidimetric method, it utilizes spectrophotometric measurement
validated to ensure there is no inhibition or enhancement. at an absorbance wavelength designated for the colorant
(p-nitroaniline). The chromogenic method utilizes initial portions
For all methodologies, sample positive product controls (PPCs)
of the endotoxin LAL reaction-cascade to activate an enzyme
are run to measure potential inhibition or enhancement in
causing the release of p-nitroaniline from the synthetic material
the sample extraction solution. There may also be additional
yielding the yellow pigment. The amount of the yellow colorant
QC requirements such as test replicates or standard curve
in a sample is derived from the standard curve to obtain a
requirements (R2 values, CV%, Y-intercept, etc.).
quantitative result.
These methods are detailed below:
Photometric methods of analysis (Turbidimetric and Chromogenic)
Gel clot are not suitable for samples with absorbance (pigment) at the
measured wavelength used for analysis.
The gel clot method of analysis is similar to how the horseshoe
crabs immune system responds to endotoxin, by clotting. The New Technologies
method of analysis involves testing dilutions of the extract and
New recombinant technologies have been developed to reduce
determining whether the solution forms a solid gel in the presence
the need for harvesting horseshoe crab blood for this testing.
of the horseshoe crab derived reagent. This method produces
These technologies currently require additional validation work
only a pass / fail result and is more subjective than the other
and submission to regulatory bodies, as alternative methods for
methods as it is a very manual test. This test is now rarely used
use in product release testing.
due to improved methods being available.

Kinetic Turbidimetric

The turbidimetric method of analysis utilizes spectrophotometry

to measure the intensity/absorbance of light passing through
a sample solution. If endotoxin is present in this method, the
horseshoe crab derived reagent produces a clotting reaction that
produces a solid mass in the solution. The cloudiness or turbidity
of the sample affects the amount of light that can pass through

F O R M O R E I N F O R M AT I O N
STERIS Applied Sterilization Technologies
Web: www.steris-ast.com // Email: [email protected]
(EMEA-APAC) +44 (0) 8456 88 99 70
(Americas) 877.783.7479

TechTip #66 | Rev 1, 3/2023

 TECHNICAL TIP

BACTERIAL ENDOTOXIN TESTING

REFERENCES

1 . ANSI/AAMI ST72, Bacterial Endotoxins – Test Methods, Routine Monitoring, and Alternatives to Batch Testing

F O R M O R E I N F O R M AT I O N
STERIS Applied Sterilization Technologies
Web: www.steris-ast.com // Email: [email protected]
(EMEA-APAC) +44 (0) 8456 88 99 70
(Americas) 877.783.7479

TechTip #66 | Rev 1, 3/2023