Protecting Health

Alternative Bacterial Endotoxin Test Methods

Horseshoe crabs have played a role in human health since the 1970s.  LAL and TAL methods have changed since the first gel clot test, however the last advancement came in the 1980s when a chromogenic substrate was added to the LAL/TAL chemistry.  All of the LAL and TAL methods require the bleeding of animals.  Global conservation and preservation of the horseshoe crab can be attained with alternative methods that can reduce or eliminate the need to use an animal for endotoxin testing.

There are alternative and sustainable methods available for those pharmaceutical, medical device, and dialysis companies willing to embrace sustainable, animal-friendly innovations in endotoxin detection.

Recombinant Cascade Reagent (rCR)
PyroSmart NextGen™ is a sustainable recombinant Cascade Reagent (rCR) that delivers the same reliable results as conventional LAL reagent and offers these additional advantages: No Animal Content (Horseshoe Crab Blood Free), Same Cascade, No Cross Reactivity With 1,3-b-D-glucans, Same Instruments, Same Preparation Steps, Meets Corporate Sustainability Objectives. As with naturally sourced LAL reagents, in the presence of endotoxin, recombinant Factor C (rFC) becomes an activated moiety which in turn activates recombinant Factor B and recombinant Proclotting Enzyme; ultimately resulting in the proteolytic cleavage of a colorless chromogenic substrate formulated with PyroSmart NextGen™. By relying on the same cascade mechanism, the response of recombinant Factor C is amplified the same way as by LAL reagents and thus the same sensitivity is achieved using this kinetic assay. Due to absence of Factor G, PyroSmart NextGen™ will not react with any 1,3-b-D-glucans and therefore will prevent glucan-derived enhancement and false positive results. For more information, visit

Recombinant Factor C (rFC)
The National University of Singapore developed and Lonza commercialized an endotoxin test that does not require horseshoe crab blood. Instead, the DNA for one of the horseshoe crab blood clotting factors, Factor C, was cloned and is manufactured recombinantly (synthetically). Recombinant Factor C (rFC), similar to the native Factor C, is activated by endotoxin. In the rFC test method, branded PyroGene™ by Lonza, activated rFC cleaves a fluorogenic substrate and the fluorescent signal is monitored and analyzed to quantitate endotoxin content. Recombinant technology is used to manufacture therapeutics. It makes sense to use similar technology to help assess the safety of products. The horseshoe crab provides some DNA to support this alternative advancement in endotoxin detection. Widespread adoption of this method could help significantly reduce the need to bleed horseshoe crabs. In areas of the world where horseshoe crabs are limited and/or declining, switching to the rFC method could help make a positive impact to the crab population. For more information, visit

Another company developing and supplying endotoxin detection assays based on rFC is Hyglos, originally a German start-up which was acquired by bioMérieux in 2016 ( In addition to the rFC assay ENDOZYME®, Hyglos applies endotoxin-binding molecules derived from bacteriophages for specific binding to the endotoxin present in the sample, with ENDOLISA®. By removing the interfering sample matrix prior to detection, the ENDOLISA® assay has revolutionized endotoxin testing of complex samples that typically inhibit detection when using conventional methods. Recently, Hyglos/bioMérieux has introduces ENDOZYME® II GO, a faster and easier to use rFC assay, reducing handling time by more than half and improving consistency.

The methods based on rFC have recently been included in the European Pharmacopoeia guidelines as valid alternatives to the LAL test. In order to be used for product release, the rFC methods must be validated and show to provide equivalent or better performance than LAL. In addition, the ENDOLISA® methodology for removing interfering factors is included in the European Pharmacopoeia chapter 5.1.10., section 9.

Monocyte Activation Test
Another method that does not require the use of horseshoe crab blood is the Monocyte Activation Test (MAT) or the In Vitro Pyrogen test.  This test method uses human blood rather than horseshoe crab blood.  The MAT method measures the release of cytokines from blood cells due to the presence of pyrogens, such as endotoxin, in the test sample.  The MAT is basically mimicking what occurs in our blood stream when it is exposed to pyrogenic substances.  The MAT has an advantage over the LAL/TAL and rFC methods as it can detect non-endotoxin pyrogens. Currently, there are no non-endotoxin pyrogen standards and to date the Monocyte Activation Test has not been able to show a reaction with an authentic non-endotoxin pyrogen.

Learn More

See our list of companies that purchase their endotoxin detection materials from sustainable and responsible manufacturers.

Looking for more on the science? Visit our Research section.