The determination of the reaction endpoint is made with dilutions from the material under test in direct comparison with parallel dilutions of a reference endotoxin, and quantities of endotoxin are expressed in defined Endotoxin Units. 

Since LAL reagents have also been formulated to be used for turbidimetric or colorimetric readings, such tests may be used if shown to comply with the requirements for alternative methods. These tests require the establishment of a standard regression curve and the endotoxin content of the test material is determined by interpolation from the curve. The procedures include incubation for a preselected time of reacting endotoxin and control solutions with LAL reagent and reading of the spectrophotometric light absorbance at suitable wavelengths. In the case of the endpoint turbidimetric: procedure, the reading is made immediately at the end of the incubation period. In the kinetic assays (turbidimetric and colorimetric), the absorbance is measured throughout the reaction period and rate values are determined from those readings. In the endpoint colorimetric procedure the reaction is arrested at the end of the preselected time by the addition of an enzyme-reaction-terminating agent prior to the readings.  

REFERENCE STANDARD AND CONTROL STANDARD ENDOTOXINS 

The reference standard endotoxin (RSE) is the USP Endotoxin Reference Standard, which has a defined potency of 10,000 USP Endotoxin Units (EU) per vial. Constitute the entire contents of 1 vial of the RSE with 5 mL of LAL Reagent Water, mix intermittently for 30 minutes, using a vortex mixer, and use this concentrate for making appropriate serial dilutions. 

Preserve concentrate in a refrigerator, for making subsequent dilutions for not more than 14 days. Mix vigorously, using a vortex mixer for not less than 3 minutes before use. Mix each dilution for not less than 30 seconds before proceeding to make the next dilution. Do not store dilutions, because of loss of activity by absorption in the absence of supporting data to the contrary. A control standard endotoxin (CSE) is an endotoxin preparation other than the RSE that has been standardized against the RSE. Standardize each new lot of CSE prior to use in the test. Calibration of a CSE in terms of the RSE must be with the specific lot of LAL reagent and the test procedure with which it is to be used. Standardization of a CSE against the RSE using an LAL reagent for the gel-clot procedure may be effected by assaying a minimum of 1 vial of the CSE and 1 vial of the RSE, as directed under Test Procedure, but using 4 replicate reaction tubes at each level of the dilution series for the RSE and 4 replicate reaction tubes similarly for each vial or aliquot of the CSE. The antilog of the difference between the mean log ₁₀ endpoint of the RSE and the mean log₁₀ endpoint of the CSE is the standardized potency of the CSE, which then is to be converted to and expressed in Endotoxin Units per ng under stated drying conditions for the CSE or in Endotoxin Units per container, whichever is appropriate. 

A suitable CSE has a potency of not less than 2 Endotoxin Units per ng and not more than 50 Endotoxin Units per ng. 

PREPARATORY TESTING  

Use an LAL reagent of confirmed label sensitivity. Treat any containers or utensils employed so as to destroy extraneous surface endotoxins that may be present, such as by heating in an oven at 250ºC or above for sufficient time. 
  
The validity of test results for bacterial endotoxins requires an adequate demonstration that specimens of the article, or of solutions, washings, or extracts thereof to which the test is to be applied do not of themselves inhibit or enhance the reaction or otherwise interfere with the test. Validation is accomplished performing the Inhibition or  Enhancement Test as described below. Appropriate negative controls are included. Validation muts be repeated if the LAL reagent source or the method of manufacture or formulation of the article is changed.  
 

Test for Confirmation of Labeled LAL Readgent Sensitivity 

Confirm the labeled sensitivity using at least one vial of the LAL reagent lot. Prepare a series of two-fold dilutions of the RSE (or CSE) to give concentrations of 2 l, l, 0.5 l, and 0.25 l where l is the labeled sensitivity of the LAL reagent in endotoxin Units per mL. Perform the test on the four standard concentrations in quadruplicate and include negative controls. The geometric mean endpoint concentration (see Calculations and interpretation) must be greater than or equal to 0.5 l and less than or equal to 2.0 l. Confirm the labeled sensitivity of each new lot of LAL reagent prior to use in the test.  

Inhibition or Enhancement Test 

Perform the test on aliquots of the specimen, or a dilution not the Maximum Valid Dilution, in which there is not detectable endotoxin. Perform the test on the specimen without added endotoxin and with endotoxin added to give final concentration of 2.0 l, l, 0.5l and 0.25l. Perform the test as directed under Test Procedure, but using not less than 4 replicate tubes for the untreated specimen and for each specimen to which endotoxin as been added. In parallel with the above, test in duplicate the same endotoxin concentrations in water and untreated negative controls. Calculate the geometric mean endpoint endotoxin concentration for the specimen as described under Calculations and Interpretation. The test is valid for the article if the geometric mean endpoint concentration in the specimen is greater than or equal to 0.5 l and less than or equal to 2.0 l. 

If the result obtained for the specimens to which endotoxin has been added is outside the specified limit, the article is unsuitable for the Bacterial Endotoxins Test. 

Repeat the test for inhibition or enhancement after neutralization, inactivation, or removal of the interfering substances or after the specimen has been diluted by a factor not exceeding The Maximum Valid Dilution. Use a dilution, not to exceed the Maximum Valid Dilution, sufficient to overcome the inhibition or enhancement of the known added  endotoxin, for subsequent assays of endotoxin in test specimens. 

If endogenous endotoxin is detectable in the untreated specimen under the conditions of the test, the article is unsuitable for the Inhibition or Enhancement Test, or,  it may be rendered suitable by removing the endotoxin present by ultrafiltration, or by appropriate dilution. Dilute the untreated specimen (as constituted where applicable, for administration or use), to a level not exceeding the maximum valid dilution, at which no endotoxin is detectable. Repeat the test for Inhibition or Enhancement using the specimens at those dilutions. 

Maximum Valid Dilution (MVD) 

Maximum Valid Dilution is appropriate to Injections or to solutions for parenteral administration in the form, constituted or diluted for administration, or where applicable, to the amount of drug by weight if the volume of the dosage form for administration could be varied. Where the endotoxin limit concentration is specified in the individual monograph in terms of volume (in EU per mL), divide the limit by l, which is the labeled sensitivity (in EU por mL) of the lysate employed in the assay, to obtain the MVD factor. Where the endotoxin limit concentration is specified in the individual monograph in terms of weight or of Units of active drug (in EU por mg or in EU per Unit), multiply the limit by the concentration (in mg per mL or in Units per mL) of the drug in the solution tested or of the drug constituted according to the label instructions, whichever is applicable, and divide the product of the multiplication by l, to obtain the MVD factor. The MVD factor so obtained is the limit dilution factor for the preparation for the test to be valid. 

TEST PROCEDURE 

In preparing for and applying the test, observe precautions in handling the specimens in order to avoid gross microbial contaminatuon. To quantify the amount of endotoxin in a specimen, an assay is performed on decreasing concentrations of specimens prepared by serial dilution. Select dilutions so that they correspond to a geometric series in which each step is greater than the next by a constant ratio. Include negative and positive controls and a positive product control. 

Use not less than 2 replicate reaction tubes at each level of the dilution series for each specimen under test. A standard endotoxin dilution series involving not less than 2 replicate reaction tubes is conducted in parallel. A set of standard endotoxin dilution series is included for each block of tubes, which may consist of a number of racks for incubation together, provided the environmental conditions within blocks are uniform. 

Preparation 

Since the form and amount per container of standatrd endotoxin and of a LAL reagent may vary, constitution and/or dilution of contents should be as directed in the labeling. The pH of the test mixture of the specimen and the LAL reagent is in the range 6.0 to 8.0 unless specifically directed otherwise in the individual monograph. The pH may be adjusted by the addition of sterile, endotoxin-free sodium hydroxide or hydrochloric acid or suitable buffers to the specimen prior to testing. 

Procedure 

Into 10- X 75-mm test tubes or single test vials, dispense the specified volumes of negative controls, standard endotoxin concentrations, specimens, and positive product controls. Positive product controls consist of the article, or of solution washing or extract thereof to which RSE, or a standardized CSE, has been added to give a concentration of 2 l. Add appropriately constituted LAL reagent, unless single test vials are used. Mix the specimen/LAL reagent mixture and place in an incubating device such as a water bath or heating block, accurately recording the time at which the tubes are so placed. Incubate each tube, undisturbed, for 60 ± 2 minutes at 37 ± l ºC, and carefully remove it for observation. A positive reaction is characterized by the formation of a firm gel that remains when inverted through 180º. Record such a result as positive (+). A negative result is characterized by the absence of such a gel or by the formation of a viscous gel that does not maintain its integrity. Record such a result as negative (-). Handle the tubes with care, and avoid subjecting them to unwanted vibrations, or false negative observations may result. The test is invalid if the positive product control is negative or the endotoxin standard does not show the endpoint concentration to be within ± 1 two-fold dilutions from the label claim sensitivity of the LAL reagent or if any negative control is positive. Proceed to Endotoxin Content Calculation to determine the amount of endotoxin present in the test specimen. 

CALCULATION AND INTERPRETATION 

Geometric Mean Calculation 

The endpoint is the last positive test in a series of decreasing concentrations of endotoxin, specimen, or specimen to which endotoxin has been added. Record the endpoint concentration, E, for each replicate series of dilutions. Determine the log endpoint concentrations, e, and calculate the geometric mean endpoint concentration using the following formula: 

where Se is the sum of the log endpoint concentrations of the dilution series used and f is the number of replicates. 

Endotoxin Content Calculation 

Calculate the concentration of endotoxin (in Units por mL or in Units per g or mg) in or on the article under test. First, calculate the endpoint concentration, E, for each of a series of dilutions by multiplying the reciprocal of each endpoint dilution factor by l, where l is the labeled sensitivity expressed in Endotoxin units por mL, of the lysate used in the test. The geometric endpoint concentration of the article under test is thus the antilog of Se/f, where e is the log of the endpoint concentration, and f is the number of replicate reaction tubes read at the level for the specimen under test. 

Interpretation 

The article meets the requirements of the test if the concentration of endotoxin is not more than that specified in the individual monograph.