Endotoxin or LAL Test
Endotoxin testing (LAL test) ensures that sterile pharmaceutical products are safe for human use.
Endotoxins are bacterial structural components that are released when such a cell is lysed. These components are toxic if administered to humans and/or animals, causing a pyrogenic response (rise in body temperature). For this reason it is important that drugs and medical devices which are either injected or implanted must be tested for their endotoxin content.
There are several methods available for conducting the endotoxin test, which includes the in vivo rabbit pyrogen test and several in vitro alternatives that utilize the Limulus Amebocyte Lysate (LAL) system.
The most common approach to endotoxin testing is the limulous amoebocyte lysate test (LAL test). This can be accomplished by various options including gel clot, kinetic chromogenic and kinetic turbidimetric assays. This methodology is also used for the evaluation of medical devices such single- use disposable equipment and implants. This is done by extracting the test product with pyrogen -free water (PFW) and testing for the presence of endotoxin in the extracts.
PRINCIPLE OF TEST:
Kinetic Chromogenic method and Kinetic Turbidimetric method
Eurofins | ams is able to perform both the kinetic chromogenic and turbidimetric assays. The chromogenic method involves an enzymatic reaction between the endotoxin and lysate which results in the production of a yellow colour in the presence of endotoxin. The intensity of the colour production is directly linked to the quantity of endotoxin present in the sample. With the kinetic variation of the assay, the time of onset of the colour reaction is measured. Therefore, with the use of endotoxin standards we are able to calculate the value of endotoxin present in or on the product. Some products are of a colour that would interfere with this form of testing, and so the turbidimetric method can be used to avoid any such interference. In this case a different lysate is used and the reaction with endotoxin results in the solution becoming turbid, thus allowing quantitation of endotoxin content without relying on the colour present. Both methods are equally effective in obtaining the endotoxin content in a product, but often, one is more suitable than the other. Both methods use objective measurements to determine endotoxin content and are quantitative in nature. These tests can be carried out relatively quickly, and results can be available within 3-5 days of sample receipt.
Gel Clot Assay method
The gel clot assay was the original LAL method and relies upon the operator to distinguish the formation of the gel clot in the reaction tubes. It is a qualitative or semi-quantitative test that is used to screen for the presence of endotoxins. A clot formation is interpreted as a positive result for the presence of endotoxin and if no clot forms, this is interpreted as the sample being endotoxin free. The results are from the subjective interpretation of the clot formation.
- Bacterial Endotoxins Test: The US Pharmacopeia. USP <85>. Current.
- Test for Bacterial Endotoxins: British Pharmacopoeia. (BP) Appendix XIV C. Current.
- Bacterial Endotoxins: European Pharmacopeia. (EP) 2.6.14. Current.
SAMPLE REQUIREMENTS AND TURNAROUND TIMES:
Validation of the inhibition or enhancement properties of the materials on the test system.
SAMPLE REQUIREMENTS [FDA and AAMI Guidelines]:
Samples from three (3) production lots should be tested for inhibition and enhancement before this test is considered validated for use with the test product. The number of samples required for each lot is the same as *below.
NOTE: Validation testing can be performed at the same time and on the same samples as the lot release (finished product) testing.
Quantitative determination of endotoxin level for finished devices or other materials.
*SAMPLE REQUIREMENTS [FDA Guidelines]:
For lots of less than 30 units – 2 sample devices
For lots of 30-100 units – 3 sample devices
For lots of 101 units or greater – 3% of lot, up
Endotoxin testing of water system samples or other liquids.
1 mL in a sealed endotoxin-free polystyrene or glass container.
Routine test results can be completed in 3 – 5 days. Depending on workloads in the laboratory, validations can be completed in 21 – 28 days.