Bacterial Endotoxins Test (BET) is use to determine the quantity of bacterial endotoxins which present in the cell wall of the gram negative bacteria. Medical devices which are subject to contact[directly/indirectly] with the lymphatic system, cardiovascular system or cerebrospinal fluid must be undergo Bacterial Endotoxins Test (BET) as part of the lot release testing.
Bacterial Endotoxins Test (BET) is mandatory for the Injectable pharmaceutical products. The validated water system which is subject to routine monitoring and the starting/incoming materials must be ensure that the final product doesn’t effect by its endotoxins. The BET is also known as LAL[Limulus Amebocyte Lysate] Test and also known as Pyrogen test due to bacterial endotoxins can cause a fever in mammals, including humans. Don’t messed up with rabbit pyrogen test which is mentioned in USP chapter <151>.
Also read the following guidance for better understanding- for Industry Pyrogen and Endotoxins
- ANSI/AAMI ST72:2011
- FDA Guidance
- EP 2.6.14
- JP 4.01
- USP Chapter <85>
- USP Chapter <161>
In pharmaceutical industry and the industry which are manufacturing sterile products/products label claim itself sterile to its intended use must be pyrogen free and BET test must be done before release of the batch/lot to the market. Sterile pharmaceutical products and Water for injections are undergo BET in a pharmaceutical firm using gel clot method.
BET is an in-vitro test which is used to seek the endotoxins presence in the specific test sample/products. Endotoxins are frequently known as Pyrogen which are mainly produced by gram-negative bacteria. The main principle of BET makes it the utmost sensitive test that one can use to detect and quantify endotoxins, toxins which are notably known for causing fever in human.
During purification, production or packaging stages of Pharmaceutical products, it can be contaminated and BET must be perform before using its as sterile parenteral solutions. To identify the presence of endotoxins, add the sample to the Lysate, an enzyme found from horse shoe crab which hemolymph cells is mainly responsible to produce Lysate. The main principle of the BET is physiological reaction between the amoebocytes and endotoxins. The amoebocytes are fond on the blood of horse shoe crabs.
Amoebocytes found in the horse shoe crabs provide protection mechanism against pathogens. The Amoebocytes contains granules possess clotting factor which is generally released when encountered by the endotoxin causing coagulation. So this is very basic that the main mechanism of BET is physiologic effect between coagulating factor and endotoxins.
At the time of BET, the combination of endotoxins and calcium, a preclotting enzyme is typically activated which play a major role to catalyze the transformation of procoagulogen into a unit generally made of polypeptide. It is marked as coagulogen, its link up by a disulfide bond form gel-clot. With the help of a spectrophotometry, the formed precipitate subject to measure to identify if there are endotoxins in a test sample.
Types of Bacterial Endotoxins Test (BET)
- Gel clot technique
- Turbidimetric method
- Chromogenic method
There are three endotoxin detection methods are available but among these methods, gel clot technique is widely used to detect the Bacterial Endotoxins which form gel that can be easily identified. Another method is known as turbidimetric method where the amount of endotoxins are measured based on the turbidity.
The selected sample introduce into a specific solution contains endogenous substrate, cleaved upon introduction of the endotoxin containing sample and generate turbidity. Intensity of the turbidity indicate the presence of endotoxin otherwise absence. The third and last method is chromogenic method which produce color. The selected sample is introduced into a solution contains synthetic complex made of peptide-chromo-gen. If the solution develop color then it indicate the presence of endotoxin to the suspected solution.
This method is very simple and time saving method. Generally 1 hour is required to determine the test result of the selected solution and more efficient compare to another method and very useful in pharmaceutical industry avoid using of animal for the similar purpose.
Procedure for Bacterial Endotoxin Test
Precautions:
Avoid touch contamination of closures.
Dehydrated endotoxin, LAL reagent and LAL water stored in a refrigerator in the temperature of (2-8)0C.
Use Endotoxin free apparatus during testing.
Preparation of Lysate:
Lysate must be reconstituted just before use by addition of the designated amount of LAL Reagent Water with the help of pipetting it directly into the vial after removing the stopper. Accumulate Lyophilized LAL powder into the bottom portion of the vial applying slight tapping on the hard surface.
Wear safety goggles if hazardous media is selected. Elude touch contamination of closures. Swirl gently but thoroughly for at 30 seconds until dissolve. Avoid any type of shaking/vibration.
Preparation of standard Endotoxin:
CSE [Control Standard Endotoxin] is an endotoxin preparation that has been standardized against the USP RSE [Reference Standard Endotoxin].Constitute the entire contents of 1 vial of the CSE with 5 ml of LAL water, mix spasmodically of 5 minutes, with the help of vortex mixture, and use this concentration to make appropriate serial dilutions.
Preserve the concentration in a refrigerator for making subsequent dilutions for 14 days only. Mix robustly with the help of vortex mixture, only for 3 minutes before use. Mix each dilution for 30 seconds before proceeding to make the next/another dilutions. Never/ever store the dilutions.
Determination of Maximum Valid Dilution (MVD):
Maximum Valid Dilution is maximum allowable dilution of a specimen/sample at which the Endotoxin Limit can be determined.
MVD applies to injection or to solution for parenteral administration in the form constituted or dilute for administration or wherever applicable, to the extent of drug by weight if the volume of dosage forms for administration could be varied.
General equation for the determination of MVD is defined as:
MVD = Endotoxin Limit × Concentration of sample solution and/ Sensitivity of reagent
When the sample under test comply with the test at a dilution less than Maximum Valid Dilution, repeat the test using greater dilution but not exceeding the MVD. The use of more sensitive Lysate permits a greater dilution of sample to be inspected.
Confirmation of labeled LAL reagent sensitivity:
Labeled sensitivity of LAL Reagent to be confirm using at least 1 vial of LAL Reagent lot. Prepare a series of two fold dilutions of the control standard endotoxin in LAL reagent water to provide concentration at 2λ, λ, 0.5 λ, 0.25 λ. Accomplish the test on four standard concentrations in quadruplicate and include the negative control.
Lysate sensitivity Test confirmation of to be carried out when a new batch of LAL Reagent is used. Mix the volume of LAL Reagent with an equal volume (such as 0.1 ml aliquots) of one of the standard solution in each test tube. Incubate reaction mixture for a constant period according to directions provided by the LAL manufacturer (usually 37±1ºC for 60±2 minutes), avoiding vibration, into incubator.
To verify/identify/test the integrity of gel, take each tube in turn directly from the incubator & invert it through about 180º in one smooth motion. If a firm/fixed gel formed which remains in place upon inversion, record the result as a positive. Mark result is negative if an intact gel is not formed/found. The test will not declare valid unless the lowest concentration of the standard solutions shows negative results in all replica test.
| Solution | Endotoxin concentration/solution to which Endotoxin is added | Number of Replicates |
| A | None / Sample solution | 2 |
| B | 2λ / Sample solution | 2 |
| C | 2λ / Water of BET | 2 |
| D | None / LAL Reagent Water | 2 |
Solution A: A sample solution of the preparation under test that is free of detectable Endotoxin
Solution B: Test for interference.
Solution C: Controlled for labeled LAL reagent Sensitivity.
Solution D: Negative control of LAL Reagent water.
Gel Clot Limit Testing:
Depyrogenate all related glassware & heat-stable materials in a hot-air oven using validated process at the time and temperature setting are 30 minutes at 250ºC respectively. Plastic apparatus like micro-plates & pipette tips for automatic pipette use only that which has been shown to be free of detectable Endotoxin and do not to interfere with test result. Perform the inhibition test on the sample dilution at dilution.
pH of the test mixture of the specimen & the LAL Reagent is in the range 6.0 to 8.0 The pH may be adjusted by the addition of sterile, Endotoxin free Sodium Hydroxide[NaOH] or Hydrochloride Acid[HCl] or Suitable Buffers to the Specimen before testing. Mix a volume of the LAL reagent with the equal volume [such as 0.1 ml aliquots] of one standard solution in each of (10 X 75) mm test tube.
Incubate reaction mixture for a constant period according to directions provided by the LAL manufacturer [usually 37±1ºC for 60±2 minutes, avoiding vibration, into incubator. To test/identify/verify the integrity of the gel, take each tube in turn directly from the incubator and invert it through about 180º in one smooth motion.
If a firm gel has formed that remains in place upon inversion, record the result as a positive. A result is negative if an intact gel is not formed. Calculate the detected Endotoxin, through used dilution factor and used LAL reagent [Lysate Sensitivity]. Generate Report the test result.
Download all annexure from below the link:
Annexure-I Endotoxin Test Report
Annexure-II Lysate Sensitivity Test Record
Annexure-III Endotoxin Test Record Logbook