Endotoxins in medical gloves for single use (EN 455-3: 2023). Quantitative Endotoxin test (European Pharmacopoeia 2.6.14: Method E – Chromogenic end-point method): LAL (Limulus Amebocyte Lysate). Chromogenic test.
Test with the Certificate of Good Laboratory Practices (GLPs).
The chromogenic LAL (Limulus Amebocyte Lysate) test is used to detect and quantify endotoxins associated with gram-negative bacteria but does not detect pyrogens related to chemicals. In the test, a synthetic substrate equivalent to the lysate of the amebocytes of the crab Limulus polyphemus is used, and it is considered the most convenient because: a) its high sensitivity; b) the results of the test are linear with the concentration of endotoxin; c) this test is recognized by the FDA, and it is recommended to guarantee that the products analyzed do not contain endotoxins (pyrogenic substances); d) in this test, a synthetic substrate linked to a chromogen is used that facilitates the reading and quantification of the reaction; e) the use of five standards in duplicate allows to elaborate a standard line for each test, to quantify the amount of endotoxin present in the sample analyzed.
According to the EN 455-3 standard, the minimum number of gloves recommended to be tested are two pairs of gloves for a batch size under thirty, three pairs of gloves for a batch size thirty to one hundred, and 3% of a batch above one hundred, up to a maximum of ten pairs of gloves per batch.
Fort the test, the outside surface of each pair of gloves is extracted with endotoxin-free water for 40 min to 60 min at a temperature between 37ºC and 40ºC ensuring that all surfaces come into contact with the extraction medium. Finally, the extract is centrifuged to remove particles, decanted and tested for endotoxin immediately.
The results are expressed endotoxin units per pair of gloves. If sterile gloves are to be labelled as “low endotoxin gloves”, the endotoxin content shall not exceed the limit of 20 endotoxin units per pair of gloves.
In our laboratory, the detection limit of the technique is 0.03 EU/mL, so for concentrations lower than this value, the result can only be reported as < 0.03 EU/mL of extract.