sop for endotoxin challenge test

sop for endotoxin challenge test


To lay down a procedure for Endotoxin Challenge Test.
2.0 SCOPE :
This SOP is applicable for Endotoxin Challenge Test in Microbiology Lab. Of Quality Control
3.1 Officer / Executive – QC (Microbiology)
4.1 Head – QC
The Endotoxin Challenge Vial (ECV) is used in the validation of dry heat depyrogenation cycles. The ability of a particular oven / Tunnel cycle to destroy / inactivate endotoxin is measured by comparing the endotoxin level in baked ECVs vs unbaked control ECVs.

6.1 material & Instruments :
6.1.1 Limulus Amebocyte Lysate Reagent
6.1.2 Endotoxin Indicator Vial (100000 EU/ml)
6.1.3 LAL Reagent Water
6.1.4 Ampoules or Vial according to requirement
6.1.5 Depyrogenated Dilution Tubes (13×100 mm)
6.1.6 Depyrogenated Assay Tubes (10×75 mm)
6.1.7 Micropipette with Pyrogen free tip (20-200 µl)
6.1.8 Micropipette with Pyrogen free tip (100-1000 µl)
6.1.9 Vortex Mixer
6.1.10 Heating Block
6.2 Preparation Of Challenge Vials :
6.2.1 Reconstitute the Challenge Vial of Endotoxin (100000 EU/ml) with 10 ml LRW to yield 10,000 EU/ml) and vortex according to manufacturer”s instructions.
6.2.2 Transfer 0.1 ml aliquot in to ampoules or vials used for Challenge Test (10000 EU/Vial).
6.2.3 Dry the ampoules or vials in Laminar Air Flow for overnight . Each ampoule or vial now contains 10000 EU/Vial. Mark the above prepared Ampoules/Vials as 1 to 10 numbers.
6.2.4 Keep at least 1 Ampoule/Vial as positive control (do not expose through Oven / Tunnel).
6.2.5 Mark the remaining ampoules/vials as NPC.
6.2.6 Expose these Ampoules/Vial to appropriate location in DHS/Tunnel as per its typical Depyrogenation Cycle.
6.3 Dilution Of Positive Control Ampoule / Vial :
6.3.1 Reconstitute the Ampoules/Vials with 1 ml LRW and vortes vigorously for 15 minutes and each subsequent dilution for 2-4 minutes.
6.3.2 Now the concentration of Endotoxin in the PPC will be 10000 EU/ml.
6.3.3 Prepare 1:20 dilution of the 10000 EU/ml to obtain 500 EU/ml.
6.3.4 Prepare 1:20 dilution of the 500 EU/ml to obtain 25 EU/ml.
6.3.5 Prepare 1:25 dilution of the 25 EU/ml to obtain 01 EU/ml.
6.3.6 From the above 1 EU/ml preparation, prepare a two fold dilution series up to 2λ, λ, λ/2, λ/4, where λ = Labelled Lysate Sensitivity, λ = 0.125 EU/ml.




1. 10000 EU/Vial 1 ml 10000 EU/ml
2. 0.1 ml of 10000 EU/ml 1.9 ml 500 EU/ml
3. 0.1 ml of 500 EU/ml 1.9 ml 25 EU/ml
4. 0.1 ml of 25 EU/ml 2.4 ml 01 EU/ml (8 λ)
5. 1 ml of 01 EU/ml 1 ml 0.5 EU ml (4 λ)
6. 1 ml of 0.5 EU/ml 1 ml 0.25 EU ml (2 λ)
7. 1 ml of 0.25 EU/ml 1 ml 0.125 EU ml (λ)
8. 1 ml of 0.125 EU/ml 1 ml 0..0625 EU ml (λ/2)
9. 1 ml of 0.0625 EU/ml 1 ml 0.0325 EU ml (λ/4)

6.4 dilution And Test Of Npc Ampoules / Vial :
6.4.1 It is assumed that three log reduction is achieved after exposure of ampoules / vials in oven /Tunnel, the Endotoxin concentration in the vial is 10 EU/Vial now.
6.4.2 Reconstitute each ampoules/Vials with 1 ml LRW and vortes vigorously for 15 minutes and the Endotoxin concentration in the each ampoule or vial is 10 EU/ml.
6.4.3 Take 0.1 ml from above each vials or ampoules of 10 EU/ml in assay tubes as duplicate and add 0.1 ml Lysate which have the potency 0.125 EU/ml.
Dilution Table

Sr. No. Endotoxin Lysate LRW Endotoxin Concentration (Eu/ml)
1. 10 EU/Vial or Ampoule Assumed 1 ml 10 EU/ml
2. 0.1 ml of 10 EU/Vial 0.1 ml 01 EU/ml

6.5 LAL Test Procedure :
6.5.1 Test the two fold dilution series prepared from the positive controls ampoules / vial in duplicate.
6.5.2 Test the 10 EU/ml dilutions prepared from each of exposed ampoules / Vial.
6.5.3 Test should be carried out in Clean depyrogenated 10×75 mm assay tubes only.
6.5.4 Protocol for Positive Control :


Sr. No. Dilutions CSE Dilution Used LRW Lysate in µl No. of Replicates
1. 100 µl of 2λ 100 µl 2
2. λ 100 µl of λ 100 µl 2
3. λ/2 100 µl of λ/2 100 µl 2
4. λ/4 100 µl of λ/4 100 µl 2
5. Negative Water Control (NWC) 100 µl 100 µl 2

6.5.5 Protocol for Negative Product Control (Challenged Ampoules/Vial):

Ampoule No. Dilutions Endotoxin Indicator Dilution (01 EU/ml) Lysate in µl No. of Replicates
1. NPC 100 µl 100 µl 2
2. NPC 100 µl 100 µl 2
3. NPC 100 µl 100 µl 2
4. NPC 100 µl 100 µl 2
5. NPC 100 µl 100 µl 2
6. NPC 100 µl 100 µl 2
7. NPC 100 µl 100 µl 2
8. NPC 100 µl 100 µl 2
9. NPC 100 µl 100 µl 2
10. NPC 100 µl 100 µl 2
11. NPC 100 µl 100 µl 2
12. NPC 100 µl 100 µl 2

6.6 Calculation :
6.6.1 Formula :
Endotoxin Concentration = Lysate sensitivity × Reconstitued Volume × Dilution
For Unbaked (Positive Control) Vials = 0.125 EU/ml × 1 ml/Vial × 10,000 = 1250 EU/ml
For Baked (Heat Treated) Vials = 0.125 EU/ml × 1 ml/Vial × 1 = 0.125 EU/ml
Calculate the minimum log reduction as follows :
Minimum Log Reduction = Log Endotoxin Concentration of the Unbaked Control – Log Endotoxin Concentration of the Baked Vials.
Minimum Log Reduction = Log value of recovered Endotoxin from positive control – Log value of recovered sample from heat treated sample.
6.7 Interpretation Of Results / Acceptance Criteria :
6.7.1 Test results are valid of recovery of Endotoxin in non exposed vials is with in a two fold dilution of the labeled claim.
6.7.2 The depyrogenation cycle is considered as successfully validated if there is more than 3 log reduction is achieved in challenge Endotoxin vials exposed in to Oven/ Tunnel at specified place. Heat Exposed Vials.
6.7.3 For a valid Depyrogenation cycle, the PPC must be positive and NPC’s must be negative indicating a greater than 3 log reduction of pyroburden.
6.7.4 Record the “ Endotoxin Challenge Test Record in Annexure –I.

SOP                 Standard Operating Procedure

No.                  Number

QA                   Quality Assurance

QC                   Quality Control

QM                  QC-Microbiology

Ltd.                  Limited

GPT                 Growth Promotion Test

LAF                 Laminar Air Flow

°C                    Degree Celsius

ml                    Milliliter

NLT                 Not Less Than

PPC                  Positive Product Control

NPC                 Negative Product Control

LRW                LAL Reagent Water

EU                   Endotoxin Unit

ml                    Mililitre

µl                     Micron litre

λ                      Sensitivity of the Lysate



Annexure  No. Title of Annexure Format No.
Annexure – I  Endotoxin Challenge Test Report


  • Master Copy           Quality Assurance Department
  • Controlled Copy No. 01 Quality Assurance
  • Controlled Copy No. 02 Quality Control (Microbiology)


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sop for endotoxin challenge test

Hold time study protocol for sterilized media

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sop for Validation protocol of steam sterilizer autoclave

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sop for Operation Calibration and Maintenance of Micropipette

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