Endotoxin is heat resistant and stable in most environmental conditions. For example, it can only be destroyed at high temperatures (185°C for 3 hours or 250°C for 30 min.); by aggressive chemicals such as NaOH or H₂O_2; orremovedbyexpensive chromatographic processes. Consequently, it can be a major problem source in contamination control.

Endotoxin control is a critical quality requirement in the manufacture of parenterals, biologicals, medical devices, and in the operation of dialysis centers, blood donor centers, and in other areas specifically regulated for endotoxin content and the prevention of endotoxin contamination.

For several decades the determination of endotoxin levels were regulated by national & international agencies. In 2001, the International Conference on Harmonization (ICH) issued a guideline for endotoxin testing which has been adopted by the major pharmacopoeias, such as the USP (United States Pharmacopoeia), EP (European Pharmacopoeia) BP (British Pharmacopoeia), and JP (Japanese Pharmacopoeia). The techniques that were accepted include:

Gel-clot limit test
Gel-clot assay
Endpoint Turbidimetric
Kinetic Turbidimetric
Endpoint Chromogenic
Kinetic Chromogenic

From a legal view-point, Gel-clot tests are defined as the standard reference method, except in cases of approved applications such as NDAs, ANDAs, etc., where other techniques can be specified as being legally valid.

Endotoxin standards set the foundation for any of the testing methodologies. The current reference standard (RSE) has been developed through international collaboration, and may be purchased from the USP as EC-6.

Secondary standards such as Control Standard Endotoxin (CSE) may be established and used in determinations provided that the standardization procedure relative to RSE is in compliance with the regulation.

Endotoxin results are expressed as EU/unit or IU/unit. These will be; EU/ml, or EU/ng. IU/ml, or IU/ng; 1 EU = 1 IU.

Special Definitions, Meanings, and Utilities

Endotograph^TM – Definition: Endotographs are transformed Gaussian functions of LAL-endotoxin kinetic reaction data,

where,

C is the concentration,
m is the x-coordinate of the inflection points (averaged over the original replicate curves),
s represents the standard deviation of the x-coordinates from the original replicate curves,
K =1 - INT(log(C_MIN)-1), if INT(log(C_MIN)) = log(C_MIN) and C_MIN < 1, or
K =1 - INT(log(C_MIN)), where C_MIN is the least concentration value during the test.
The relationship between the maximum of the transformed Gaussian-function (y_MAX) and the concentration (C) will be:

Endotograph^TM – Meaning: Endot