Bacterial Endotoxin Test Methods

The test for bacterial endotoxins (BET) measures the concentration of bacterial endotoxins that may be present in the sample or on the article to which the test is applied using a lysate derived from the hemolymph cells or amoebocytes of the horseshoe crab, Limulus polyphemus. Other species of horseshoe crab namely Tachypleus gigas, Tachypleus tridentatus and Carcinoscropius rotundicauda also yield amoebocyte lysate having similar activity.

The addition of a solution containing endotoxins to a solution of the lysate produces turbidity, precipitation or gelation of the mixture. However, the addition of a chromogenic substrate to a solution of the lysate results in the development of color due to the release of chromophore from the substrate upon activation by the endotoxin present in the solution. The rate of reaction depends on the concentration of endotoxin, the pH and the temperature. The reaction requires the presence of certain bivalent cations, a clotting cascade enzyme system and clot able protein, all of which are provided by the lysate.

The quantities of endotoxins are expressed in defined Endotoxin Units (EU). With the adoption of the second International Standard for endotoxin by the Expert Committee on Biological Standards of the World Health Organization, 1EU= 1 IU.

The endotoxin limit for a given test preparation is calculated from the expression K/M, where M is the maximum dose administered to an adult (taken as 70 kg for this purpose) per kg per hour. The value of K is 5.0 EU/kg for parenteral preparations except those administered intrathecally and is 0.2 EU/kg for preparations intended to be administered intrathecally.

The test should be carried out in a manner that avoids microbial contamination. If necessary, the containers should be treated to eliminate surface endotoxins that may be present by heating in an oven at 250° before carrying out the test for endotoxins in the preparation under examination it is necessary to verify
(a) in the case of gel-clot methods, the sensitivity of the lysate;
(b) in the case of quantitative methods, the linearity of the standard curve;
(c) the absence of interfering factors, which inhibit or enhance the reaction or otherwise interfere with the test on the preparation under examination;
(d) the adequacy of the containers to resist adsorption of endotoxins.
Related: Bacterial Endotoxin Test (BET or LAL Test) Validation

Special Reagents

Endotoxin reference standard and control standard endotoxin.

• The Endotoxin Reference Standard (ERS) is the freeze-dried, purified endotoxin of Escherichia coli, which is calibrated in Endotoxin Units (EU) by comparison with the International Standard.
• The Endotoxin Reference Standard (ERS) or any other suitable preparation the activity of which has been determined in relation to the ERS or the International Standard using a gel-clot or other suitable method.
• The freeze-dried endotoxin should be reconstituted with water BET by mixing intermittently for 30 minutes using a vortex mixer. The concentrate should be stored in a refrigerator for not more than 28 days. Subsequent dilutions of the concentrate should be made by mixing vigorously for not less than 3 minutes before use. Each dilution should be mixed for not less than 30 seconds before proceeding to make the next dilution.
• A Control Standard Endotoxin (CSE) which is suitably standardized against the ERS may be used for routine bacterial endotoxin testing.

Lysate

A lysate of amoebocytes from either of the species of the horseshoe crab, Limulus polyphemus, Tachypleus gigas, Tachypleus tridentatus or Carcinoscorpius rotundicauda reconstituted as stated on the label. The species from which the lysate is obtained is stated on the label.

Water BET

Water that gives a negative result under the conditions prescribed in the test for bacterial endotoxins on the preparation under examination. It may be prepared by distilling water three times in an apparatus fitted with an effective device to prevent the entrapment of droplets or by other means that give water of the desired quality.
0.1 M Hydrochloric acid BET: Prepare from hydrochloric acid using water BET. After adjustment of the pH 6.0 to 8.0 with 0.lM sodium hydroxide BET it gives a negative result under the conditions of the test.
0.1 M Sodium hydroxide BET: Prepare from sodium hydroxide using water BET. After adjustment of the pH to 6.0 to 8.0 with 0.1M hydrochloric acid BET it gives a negative result under the conditions of the test.
Tris-chloride buffer pH 7.4 BET: Dissolve 0.6057 g of tris (hydroxymethyl) methylamine in 30 ml of water BET, add 0.33 ml of hydrochloric acid, dilute to 100 ml with water BET and mix. It gives a negative result under the conditions of the test.
NOTE: Special reagents used in the test may use the suffix 'LAL', 'TAL' or TAL', as the case may be, to indicate the species of the horseshoe crab from which the amoebocyte lysate is derived. They have the same significance as the suffix 'BET'.

The following methods can be used to monitor the endotoxin concentration in a product official in the Pharmacopoeia and to determine whether the product complies with the limit specified in the monograph.
Also see:
Gel-Clot Methods
Quantitative Methods