LAL Endotoxin Testing

What is Endotoxin Testing?

LAL (Limulus amebocyte lysate) endotoxin testing is an in-vitro assay used for the detection and quantitation of bacterial endotoxins in injectable products or implantable medical devices that will make direct or indirect contact with the bloodstream or spinal fluid. This test is also used in monitoring water systems and raw materials to ensure that this does not contribute endotoxins to the finished product.

Endotoxin Testing and Why it is Important

Endotoxin is a lipopolysaccharide found in the cell wall of gram-negative bacteria and is a type of pyrogen (fever-causing agent). If endotoxin is present in injectable pharmaceuticals or on implantable medical devices and meets the bloodstream or spinal fluid, then it can cause a cascade effect of events starting with fever, septic shock, organ failure and potentially death. This is the reason that it is important to perform LAL endotoxin testing of these products as required by pharmaceutical compendia or FDA regulations to ensure a safe product for patient use.

How is Endotoxin Detected or How Does the Method Work?

The presence of bacterial endotoxin is detected in-vitro by using LAL, which comes from the blood cells of horseshoe crabs. The lysate forms a gel clot in the presence of endotoxin thus resulting in a simple and sensitive assay.

Primary Methods for Detection

As indicated in the USP <85>, there are three primary methods for detection of Endotoxin that are used in the pharmaceutical and medical device industry:

1. Gel Clot Method: This is a qualitative method based on a gel clot formation when endotoxin interacts with Limulus amebocyte lysate (LAL).
2. Turbidimetric Method: This method is based on the development of turbidity after cleavage of an endogenous substrate.
3. Kinetic/Chromogenic Method: This method is based on the development of color after cleavage of a synthetic peptide-chromogen complex.

There are newer rapid systems currently on the market that are based on the methods indicated above that provide rapid and quantitative results.

In the event of doubt or dispute, the final decision is made based upon the gel-clot limit test unless otherwise indicated in the monograph for the product being tested. The test is carried out in a manner that avoids endotoxin contamination.

Inhibition/Enhancement Requirements for the Methods

Testing is also required to be performed for the test samples to make sure the samples do not interfere (inhibit or enhance) with the ability of the method to detect endotoxins. It is recommended to perform this testing on 3 lots of the sample.

Reference Methods and Guidance for LAL Endotoxin Testing

• USP <85>
• USP <161>
• USP <797>
• AAMI ST72
• EP 2.6.14
• JP 4.01
• FDA Guidance for Industry Pyrogen and Endotoxins Testing: Questions and Answer