SOP For Endotoxin Challenge Test

PHARMA SCHOLARS
MICROBIOLOGY DEPARTMENT
STANDARD OPERATING PROCEDURE

Restricted Circulation
Department: Quality Control
TITLE: ENDOTOXIN CHALLENGE TEST
SOP No. testing areas Revision No.
Effective Date Supersedes No.
Review Date Page No. 1 of 12
1.0 OBJECTIVE :
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
Department of ……………………...
3.0 RESPONSIBILITY:
3.1 Officer/Executive – QC (Microbiology)
4.0 ACCOUNTABILITY:
4.1 Head - QC
5.0 DEFINITION:
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.
6.0 PROCEDURE:
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Officer/Executive Department Head Head Quality Assurance
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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 (12,50,000 EU/VIAL) with 5 ml LRW to yield 2,50,000
EU/ml) and vortex according to manufacturer”s instructions.
6.2.2 Transfer whole 5 ml content of Challenge Vial (2,50,000 EU/ml) in to 20 ml vial and make up the
volume 12.5 ml with LRW according to factor (1:2.5) and get the Challenge Vial of 1,00000 EU/ml
(Challenge Indicator).
6.2.3 Transfer 0.1 ml aliquot in to ampoules or vials used for Challenge Test (10000 EU/Vial).
6.2.4 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.
6.2.5 Keep at least 1 Ampoule/Vial as positive control (do not expose through Oven / Tunnel).
6.2.6 Mark the remaining ampoules/vials as NPC.

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6.2.7 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 vortex 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.
6.3.6 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.
DILUTION TABLE
S.No. ENDOTOXIN LRW ENDOTOXIN CONCENTRATION (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)

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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 vortex 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
S.No. ENDOTOXIN LYSATE LRW ENDOTOXIN CONCENTRATION

(EU/ML)
1. 10 EU/Vial or Ampoule - 1 ml 10 EU/ml

Assumed
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.

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6.5.4 Protocol for Positive Control :
S.No. Dilutions CSE Dilution Used LRW Lysate in µl No. of Replicates
1. 2λ 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 - 100 µl 100 µl 2

Control (NWC)
6.5.5 Protocol for Negative Product Control (Challenged Ampoules/Vial):
Ampoule Dilutions Endotoxin Indicator Lysate in µl No. of Replicates

No. Dilution (01 EU/ml)
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

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9. 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.
OR
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.

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7.0 ABBREVIATION:
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

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8.0 ANNEXUREURE :
Annexure No. Title of Annexure Format No.

Annexure - I Endotoxin Challenge Test Report F01-00
9.0 DISTRIBUTION :
• Master Copy Quality Assurance Department
• Controlled Copy No. 01 Quality Assurance
• Controlled Copy No. 02 Quality Control (Microbiology)
10.0 REFERENCES :
• IH
11.0 REVISION HISTORY :
Revision No. Change Control Details of Reason of Effective Done By

No. Changes Changes Date
00 Not Applicable Not Applicable New SOP

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ANNEXURE-I
PHARMA SCHOLARS
ENDOTOXIN CHALLENGE TEST REPORT
Date of Vial Exposure No. of Exposed Vial

Date of Testing Date of Release
Tunnel / Oven Location Tunnel / Oven ID No.
Performed By Date of Analysis
Shift BET Kit Manufacturer
Heating Block Temperature 37°C ± 1°C Incubation Time 60 Minutes ± 2 Minutes
Incubation Started Date Incubation Completed
at
REAGENT DETAILS :
REAGENT DETAILS LIMULUS CONTROL LAL REAGENT

AMOEBOCYTES STANDARD WATER (LRW)
LYSATE (LAL) ENDOTOXIN (CSE)
Lot No.
Expiry Date
Sensitivity/Potency
Date of Reconstitution/Opening
Use Before
Manufacturer

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DILUTION PREPARATION FOR HEAT TREATED VIAL (10 EU/VIAL ASSUMED):
Test Endotoxin
S.No. Test LRW Lysate
Dilution Concentration
- - 10 EU/Vial or Ampoule Assumed 1 ml - 10 EU/ml
1. - 0.1 ml of 10 EU/ml - 0.1 ml 01 EU/ml
DILUTION PREPARATION FOR POSITIVE CONTROL VIAL ( 10,000 EU/VIAL) :
Endotoxin
S.No. Test Dilution Test LRW
Concentration
- - 10,000 EU/Vial or Ampoule 1 ml 10,000 EU/ml
1. 1:10 0.1 ml of 10,000 EU/ml 0.9 ml 500 EU/ml
2. 1:10 0.1 ml of 500 EU/ml 0.9 ml 25 EU/ml
3. 1:25 0.1 ml of 25 EU/ml 2.4 ml 01 EU/ml (8 λ)
4. 1:2 1 ml of 01 EU/ml 1 ml 0.5 EU ml (4 λ)
5. 1:2 1 ml of 0.5 EU/ml 1 ml 0.25 EU ml (2 λ)
6. 1:2 1 ml of 0.25 EU/ml 1 ml 0.125 EU ml (λ)
7. 1:2 1 ml of 0.125 EU/ml 1 ml 0.0625 EU ml (λ/2)
8. 1:2 1 ml of 0.0625 EU/ml 1 ml 0.0325 EU ml (λ/4)

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PREPARATION OF TEST SOLUTION:
Solution Tube No. Product Quantity LRW LAL Reagent Total Volume
01 100 µl - 100 µl 200 µl
NPC
02 100 µl - 100 µl 200 µl
01 - 100 µl 100 µl 200 µl
NWC
02 - 100 µl 100 µl 200 µl
OBSERVATION:
RESULT OF NEGATIVE PRODUCT CONTROL & NEGATIVE WATER CONTROL:
Observation Vial No.

- Tube No.
1 2 3 4 5 6 7 8 9
01
NPC
02
01
NWC
02

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RESULTS OF POSITIVE CONTROL:
Endotoxin Dilution Negative Water

Tube No.
2λ λ λ/2 λ/4 Control
01
02
+ve : Gel formation -ve : No Gel formation

Calculate the minimum log reduction as follows :
Minimum Log Reduction = Log Endotoxin Concentration of the Unbaked Control – Log Endotoxin
Concentration of the Baked Vials.
OR
Minimum Log Reduction = Log value of recovered Endotoxin from positive control – Log value of
recovered sample from heat treated sample.
Endotoxin Concentration =
Minimum Log Reduction =
Remark: The depyrogenation cycle of Oven/Tunnel complies / does not comply of three log reduction for
Endotoxin Challenge Test.
Microbiologist: Checked By:

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SOP For Endotoxin Challenge Test

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PHARMA SCHOLARS

STANDARD OPERATING PROCEDURE

3.1 Officer/Executive – QC (Microbiology)

--- Prepared By Checked By Approved By

FORMAT No.: …………………………..

STANDARD OPERATING PROCEDURE

6.1 MATERIAL & INSTRUMENTS :

--- Prepared By Checked By Approved By

FORMAT No.: …………………………..

STANDARD OPERATING PROCEDURE

S.No. ENDOTOXIN LRW ENDOTOXIN CONCENTRATION (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)

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)

--- Prepared By Checked By Approved By

FORMAT No.: …………………………..

STANDARD OPERATING PROCEDURE

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:

S.No. ENDOTOXIN LYSATE LRW ENDOTOXIN CONCENTRATION

1. 10 EU/Vial or Ampoule - 1 ml 10 EU/ml

2. 0.1 ml of 10 EU/Vial 0.1 ml - 01 EU/ml

6.5 LAL TEST PROCEDURE :

--- Prepared By Checked By Approved By

FORMAT No.: …………………………..

STANDARD OPERATING PROCEDURE

6.5.4 Protocol for Positive Control :

S.No. Dilutions CSE Dilution Used LRW Lysate in µl No. of Replicates

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 - 100 µl 100 µl 2

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

Ampoule Dilutions Endotoxin Indicator Lysate in µl No. of Replicates

1. NPC 100 µl 100 µl 2

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FORMAT No.: …………………………..

STANDARD OPERATING PROCEDURE

9. NPC 100 µl 100 µl 2

--- Prepared By Checked By Approved By

FORMAT No.: …………………………..

STANDARD OPERATING PROCEDURE

SOP Standard Operating Procedure

--- Prepared By Checked By Approved By

FORMAT No.: …………………………..

STANDARD OPERATING PROCEDURE

Annexure No. Title of Annexure Format No.

11.0 REVISION HISTORY :

Revision No. Change Control Details of Reason of Effective Done By

--- Prepared By Checked By Approved By

FORMAT No.: …………………………..

STANDARD OPERATING PROCEDURE

Date of Vial Exposure No. of Exposed Vial

REAGENT DETAILS LIMULUS CONTROL LAL REAGENT

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FORMAT No.: …………………………..

STANDARD OPERATING PROCEDURE

DILUTION PREPARATION FOR HEAT TREATED VIAL (10 EU/VIAL ASSUMED):

DILUTION PREPARATION FOR POSITIVE CONTROL VIAL ( 10,000 EU/VIAL) :