The Bacterial Endotoxins Test (BET) detects or quantifies endotoxins from gram-negative bacteria using amoebocyte lysate from the horseshoe crab (Limulus polyphemus or Tachypleus tridentatus).
Endotoxins are components of gram-negative bacteria’s outer cell membrane. If these endotoxins are injected into humans or animals, they can cause multiple biological effects.
The presence of endotoxins in raw materials and parenteral products is likely due to the ubiquitous nature of gram-negative bacteria.
To ensure the safety of humans, drugs meant for parenteral administration must be free from endotoxins. Therefore, endotoxins must be controlled to a specific limit. The method to control endotoxins is called the Bacterial Endotoxin Test (BET). This method is capable of controlling resident endotoxins based on a product-specific, dose-dependent, route of administration-dependent, and time of administration-dependent calculation.
Let’s understand the key parameters about what are endotoxins, pyrogens, biological and endotoxin indicators.
Endotoxins vs Pyrogens
Endotoxins are fever-inducing substances found in the cell wall of certain gram-negative bacteria.
Pyrogens are substances that can cause fever when injected or infected.
Bacterial Endotoxin Test (BET) is an assay for measuring active endotoxin by combining a liquid test sample with Limulus amebocyte lysate (LAL) reagent and measuring the resulting proportional reaction via visual, turbidimetric, chromogenic, or other validated means of detection.
Biological Indicator vs Endotoxin Indicator
A test system containing pure and specified microorganisms with defined resistance to a specific sterilization process.
An article was tested with a vial containing endotoxin or a carrier spiked with endotoxin. The purpose of the test was to study depyrogenation. The endotoxin used in the test was a purified lipopolysaccharide and was validated to be used on an endotoxin indicator. The carrier used was made from a material suitable for the depyrogenation processes it would undergo. The endotoxin was added to the carrier at a specific concentration.
The formula for calculating an endotoxin limit in parenteral is generally noted as
Bacterial Endotoxin Test (BET) = K/M
where K is the threshold pyrogenic dose and M is the dose of the drug in units/kg/hr.
Let’s understand more about K and M
“K”, the numerator of the endotoxin limit formula, is the Threshold Pyrogenic Dose (TPD).
This is a constant, and represents a statistical assessment of the levels of endotoxin activity that it takes to induce a fever in a rabbit.
For any products administered intravenously (IV) or intramuscularly (IM), 5 EU/kg/hr is the TPD.
For products with a dose expressed per square meter of body surface, the term K is an amount of endotoxin per square meter.
For a product administered intrathecally (IT), meaning the material is injected into the space that holds cerebrospinal fluid, K is lowered to 0.2 EU/kg/hr.
If the drug product is a radiopharmaceutical, K is 175 EU for non-intrathecal routes of administration and 14 EU for intrathecal administration.
M = Dose/kg/hr
“M”, the denominator in the endotoxin limit formula, is the maximum dose of the material per kg of patient and per hour of total administration.
The dose of the material is historically interpreted to mean the dose of the active ingredient unless the product is administered on a volume/kg basis.
When the product is to be injected at frequent intervals or infused continuously, M becomes the maximum total dose administered in a single hour period. It is often desirable to express the endotoxin limit in EU/mL because these are the units of the endotoxin standards used in endotoxin testing.
K and M Values
From the literature and standards, the target patient population is adults, the average adult in the US weighs 70kg. The maximum dose/kg/hr is generally the largest dose given to the smallest patient in the target group. If the largest dose/kg/hr is the paediatric dose for a given drug product, then it is used as M.
Bacterial Endotoxin Test (BET) limit Calculation
The calculation of the endotoxin limits needs minimum of three different information that is vital to derive at a correct limit.
- Route of administration
- Dose per kilogram of patient
- Dose per hour
Based on the learning above, for the IV and IM administration, K will be 5 EU/kg and for intrathecal administration K will be 0.2 EU/kg.
If the dose is given as a “per person” then it must be adjusted by dividing the dose by the weight of the target population to get dose/kg.
For E.g., 700mg per person, then 700/70 = 10mg/Kg
If the same whole person dose applies to both adults and children, the paediatric dose/kg/hr will be higher than the adult dose/kg/hr.
If the total dose is administered by an infusion lasting “n” hours, it must be adjusted by dividing the total dose by the length of the administration to get dose/hour.
For E.g., 700mg per person infused over 7 hrs, then 700/7 = 100mg/hr
For any given route of administration, the endotoxin limit is inversely proportional to the dose. The lower the dose, the higher the limit per unit dose.
- If the dose is 1 mg/kg/hr, the limit is (5 EU/kg/hr) ÷ (1 mg/kg/hr) = 5 EU/mg
- If the dose is 10 mg/kg/hr, the limit is (5 EU/kg/hr) ÷ (10 mg/kg/hr) = 0.5 EU/mg
- If the dose is 100mg/kg/hr, the limit is (5 EU/kg/hr) ÷ (100 mg/kg/hr) = 0.05 EU/mg
Based on the standard practice, the limit less than or equal to the stated concentration is acceptable however USP <85> general chapter recommends the article must be less than the proposed limit and a sample containing a concentration equal to the limit fails the test (as does a concentration that exceeds the limit).
Bacterial Endotoxin Test (BET) Control for Speciality Products
For ophthalmic irrigation products, for which the limit is not more than (NMT) 0.5 EU/mL, and injected or implanted drug products, which the limits are for NMT 2.0 EU/dose/eye.
The chapter states that the BET is typically not required for topically applied ophthalmic products and that it does not apply to medical devices that are injected or implanted.
The general endotoxin limits of 20 EU/device for most devices that are labelled as non-pyrogenic and 2.15 EU/device for devices that contact the cerebrospinal fluid (CSF).
As an aqueous solution is required for the BET, most medical devices have to be rinsed or extracted to obtain a test solution. The USP chapter gives a formula for the calculating an endotoxin limit specific to the extraction volumes.
Endotoxin limit = K x N/V
K = the endotoxin limit per device (for example, 20 EU/device – see above)
N = is the number of devices to be tested
V = is the total volume of extract or rinse (i.e. extract volume per device multiplied by the number of extracts pooled).
Methods for Bacterial Endotoxin Test (BET) Measurement
Generally, Endotoxins can be measured using 3 techniques.
- the gel-clot technique
- the turbidimetric technique
- the chromogenic technique
The principals involved in the technique are the gel-clot technique is based on gel formation, the turbidimetric technique, based on the development of turbidity after cleavage of an endogenous substrate, and the chromogenic technique based on the development of colour after cleavage of a synthetic peptide-chromogen complex.
In case of any dispute in the results, the final decision should be made based on the gel-clot limit test unless otherwise indicated in the monograph for the product.
Monograph general chapters classify the methods in to kinetic and end point determinations that further delineated the 3 techniques.
Method A. Gel-clot method: limit test
Method B. Gel-clot method: quantitative test
Method C. Turbidimetric kinetic method
Method D. Chromogenic kinetic method
Method E. Chromogenic end-point method
Method F. Turbidimetric end-point method
1. TURBIDIMETRIC TECHNIQUE (METHODS C AND F)
This technique is a photometric test to measure the increase in turbidity. Based on the test principle employed, this technique may be classified as being either the end-point-turbidimetric test or the kinetic-turbidimetric test.
The end-point-turbidimetric test (Method F) is based on the quantitative relationship between the endotoxin concentration and the turbidity (absorbance or transmission) of the reaction mixture at the end of an incubation period.
The kinetic-turbidimetric test (Method C) is a method to measure either the time (onset time) needed for the reaction mixture to reach a predetermined absorbance or transmission, or the rate of turbidity development.
The test is carried out at the incubation temperature recommended by the lysate manufacturer (usually 37 ± 1 °C).
2. CHROMOGENIC TECHNIQUE (METHODS D AND E)
This technique is used to measure the chromophore released from a suitable chromogenic peptide by the reaction of endotoxins with the lysate. Depending on the test principle employed, this technique may be classified as being either the end-point-chromogenic test or the kinetic-chromogenic test.
The end-point-chromogenic test (Method E) is based on the quantitative relationship between the endotoxin concentration and the quantity of chromophore released at the end of an incubation period.
The kinetic-chromogenic test (Method D) measures either the time (onset time) needed for the reaction mixture to reach a predetermined absorbance, or the rate of colour development. The test is carried out at the incubation temperature recommended by the lysate manufacturer (usually 37 ± 1 °C).