REVITAL HEALTHCARE (EPZ) LTD.

T I T L E :SOP FOR BACTERIAL ENDOTOXIN TEST (LAL TEST)

PROCEDURE NO.: ISSUE DATE: EFFECTIVE DATE: NEXT REVIEW DATE:

RHEL/QA/SOP/160 01/02/2021 01/02/2021 01/02/2023

REVISION NO.:00

BACTERIAL ENDOTOXINS TEST

PROCEDURE

APPROVED BY: SIGN & DATE

Mariannah Kimatu
(QC-Microbiologist)
(Prepared By)

Nancy Mwangi
(Ass. Quality Assurance Manager)
(Reviewed By)  
 
Kevin Mochama
(Quality Control Manager)
(Reviewed By)
 
Lucycaster Muoti
(Quality Assurance Manager)
(Approved By)  

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 REVITAL HEALTHCARE (EPZ) LTD.
T I T L E :SOP FOR BACTERIAL ENDOTOXIN TEST (LAL TEST)

PROCEDURE NO.: ISSUE DATE: EFFECTIVE DATE: NEXT REVIEW DATE:

RHEL/QA/SOP/160 01/02/2021 01/02/2021 01/02/2023

REVISION NO.:00

REVISION HISTORY
Revision No. Revised Page(s) Description Date
00 Original issue 01/02/2021

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 REVITAL HEALTHCARE (EPZ) LTD.
T I T L E :SOP FOR BACTERIAL ENDOTOXIN TEST (LAL TEST)

PROCEDURE NO.: ISSUE DATE: EFFECTIVE DATE: NEXT REVIEW DATE:

RHEL/QA/SOP/160 01/02/2021 01/02/2021 01/02/2023

REVISION NO.:00

PURPOSE:
To perform Bacterial Endotoxin (LAL) test for detection and/or quantifying of endotoxins for Gram-
negative bacteria on sterile products or material to be tested.

SCOPE:
To assure the sterility level on the sterile product.

RESPONSIBILITIES:
QC, Microbiologist – To carry out the Bacterial Endotoxin Test in Microbiology Laboratory

ACCOUNTABILITY:
HOD – to ensure that the BET test is carried out once per month.

DEFINATIONS:
i. Maximum valid dilution (MVD) - is the maximum allowable dilution of a specimen at which
the endotoxin limit can be determined.
ii. Amoebocyte Lysate - lyophilized product obtained from the lysate of amoebocytes (white blood
cells) from the horseshoe crab (Limulus polyphemus or Tachypleus.
iii. Endotoxin limit - the approximate threshold of pyrogens represented in; mg/ml in the case of
endotoxin limit specified by weight (IU/mg) - units/ml in the case of endotoxin limit specified
by unit of biological activity (IU/Unit) - ml/ml when the endotoxin limit is specified by volume
(IU/ml)
iv. Lysate Sensitivity - measured optical density against known standard endotoxin concentration
expressed in EU/ml or IU/ml

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 REVITAL HEALTHCARE (EPZ) LTD.
T I T L E :SOP FOR BACTERIAL ENDOTOXIN TEST (LAL TEST)

PROCEDURE NO.: ISSUE DATE: EFFECTIVE DATE: NEXT REVIEW DATE:

RHEL/QA/SOP/160 01/02/2021 01/02/2021 01/02/2023

REVISION NO.:00

PROCEDURE:
MATERIAL REQUIREMENTS:
 Amoebocyte Lysate
 Water for Bacterial Endotoxins Test (BET)
 Endotoxin standard
 Vortex mixer
 Glass pippetes depyrogenated
 Stopwatch
 Heating block
 Clean bench
 Test tubes (for assay) depyrogenated: 10×75mm
 Test tubes(for dilutions)depyrogenated: 20×150mm
 Alluminium foil depyrogenated
 Hot air oven
 Test tube stands
 Medical device:10pcs of each product
 Sterile Disposable Gloves
 70% Isopropyl alcohol

GENERAL

 Clean all the required glassware and depyrogenate in a hot-air oven at a temperature of 250°C
for 30 minutes at 250°C.
 Transfer all the depyrogenated glassware to Microbiology laboratory (BET room).
 The use of sterile gloves and related accessory testing is required.
 Switch on the heating block as per the SOP of operating instruction for heating block and set the
temperature at 37±10C.
 Calculate the MVD for sample specimen by the following formula
MVD= Endotoxin limit× potency of the product
λ Lysate Sensitivity
MVD = (endotoxin limit × concentration of Sample Solution/(λ)
 Dilute the sample to be tested to its one- fourth MVD with LWR and vortex for 3minutes.

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 REVITAL HEALTHCARE (EPZ) LTD.
T I T L E :SOP FOR BACTERIAL ENDOTOXIN TEST (LAL TEST)

PROCEDURE NO.: ISSUE DATE: EFFECTIVE DATE: NEXT REVIEW DATE:

RHEL/QA/SOP/160 01/02/2021 01/02/2021 01/02/2023

REVISION NO.:00

Lysate Sensitivity confirmation.

Confirm in four replicates the labeled sensitivity l, expressed in EU/ml or IU/ml, of the lysate solution
prior to use in the test. Confirmation of the lysate sensitivity must be carried out when a new batch of
lysate is used or when there is any change in the experimental conditions which may affect the outcome
of the test.

Sampling and testing for water for injection

Collect the water for injection sample in depyrogenated vials or test tubes as per the sampling plan.
Testing water for injection is to be done per the method of analysis.

Preparation of the standard endotoxin stock solution

 The standard endotoxin stock solution is prepared from an endotoxin reference standard that has
been calibrated against the International Standard, for example endotoxin standard BRP.
Endotoxin is expressed in International Units (IU).
 The equivalence in IU of the International Standard is stated by the World Health Organization.
NOTE: One International Unit (IU) of endotoxin is equal to one Endotoxin Unit (E.U.). Follow
the specifications in the package leaflet and on the label for preparation and storage of the
standard endotoxin stock solution.

Preparation of the standard endotoxin solutions

 After vigorously mixing the standard endotoxin stock solution, prepare appropriate serial
dilutions of this solution using water for bacterial endotoxins test (water for BET).
 Use the solutions as soon as possible to avoid loss of activity by adsorption.

Preparation of the test solutions

 Prepare the test solutions by rinsing medical devices using water for BET.
 If necessary, adjust the pH of the test solution (or dilution thereof) so that the pH of the mixture
of the lysate and test solution falls within the pH range specified by the lysate manufacturer.
 This usually applies to a product with a pH in the range of 6.0 to 8.0. The pH may be adjusted by
the use of acid, base or a suitable buffer, as recommended by the lysate manufacturer.
 Acids and bases may be prepared from concentrates or solids with water for BET in containers
free of detectable endotoxin. Buffers must be validated to be free of detectable endotoxin and
interfering factors.

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 REVITAL HEALTHCARE (EPZ) LTD.
T I T L E :SOP FOR BACTERIAL ENDOTOXIN TEST (LAL TEST)

PROCEDURE NO.: ISSUE DATE: EFFECTIVE DATE: NEXT REVIEW DATE:

RHEL/QA/SOP/160 01/02/2021 01/02/2021 01/02/2023

REVISION NO.:00

Test procedure
Prepare solution A, B, C and D as shown in table 1.

Solution Solution LRW in µl 4 λ(CSE in Product at Lysate in µl No of

description µl) MVD/4 in replicates
µl
A Negative 50 - 50 100 2
product
control
(NPC)
B Positive - 50 50 100 2
product
control
(PPC)
C Positive 50 50 - 100 2
water
control
(PWC)
D Negative 100 - - 100 2
water
control
(NWC)

 Take 8 depyrogenated assay tubes and label the tubes by numbering and arrange in stand 1
opposite to each other i.e. stand 1&2 for NPC 3&4 for PPC 5&6 for PWC 7&8 for negative
control.
 Add 50ml of LRW in NPC and PWC, and 100ml in NWC.
 Immediately add 50ml of the product sample which is diluted at MVD/4 in a NPC and PPC, and
then add 50ml of CSE that is diluted to 4 λ In a PPC and PWC
 Finally add 100ml of lysate in all tubes and next mix the assay tubes by hands and incubate in
the heating block where the temperature is maintained at 37±10c for 60±2minutes.

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 REVITAL HEALTHCARE (EPZ) LTD.
T I T L E :SOP FOR BACTERIAL ENDOTOXIN TEST (LAL TEST)

PROCEDURE NO.: ISSUE DATE: EFFECTIVE DATE: NEXT REVIEW DATE:

RHEL/QA/SOP/160 01/02/2021 01/02/2021 01/02/2023

REVISION NO.:00

Interpretation of the results

 Each tube is interpreted as either negative or positive, the positive test indicates the formation of
firm gel capable of maintaining its integrity when the test tube is inverted 1800
 A negative test is characterized by the absence of gel or by the formation of viscous mass, which
does not hold when the tube is inverted at 1800
 The test is not valid unless both replicates of the two positive control solution B and C are
positive and those of the negative control solution D are negative.
 The preparation being examined complies with the test when a negative result is found for both
replicates of solution A and it does not comply when a positive result is found for both
replicates.
 Repeat the test if a positive result is found for one replicate of solution A and a negative result is
found in the other

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 REVITAL HEALTHCARE (EPZ) LTD.
T I T L E :SOP FOR BACTERIAL ENDOTOXIN TEST (LAL TEST)

PROCEDURE NO.: ISSUE DATE: EFFECTIVE DATE: NEXT REVIEW DATE:

RHEL/QA/SOP/160 01/02/2021 01/02/2021 01/02/2023

REVISION NO.:00

ABBREVIATIONS

SOP: standard operating procedure

QA: Quality Assurance

QC: Quality Control

BET: Bacterial Endotoxin Test

CSE: Control Standard Endotoxin

MVD: Maximum Valid Dilution

LRW: LAL Reagent Water

PPC: Positive Product Control

NPC: Negative Product Control

PWC: Positive Water Control

NWC: Negative Water Control

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