2019/10/24

Confidential

Endotoxin Indicator

October 2019

What’s Endotoxin Indicator ?

Endotoxin Indicator is intended for performing validation

for depyrogenation processes of dry heat oven cycles.

A product used to scientifically verify and guarantee

a dry heat sterilizer ( or other cleaning equipment)
has the ability to depyrogenate.

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Components of Endotoxin indicator

 Endotoxin indicator contains a purified

lipopolysaccharide (E.Coli 055:B5) that is
known a endotoxin concentration designed to
validate depyrogenation process.

 This product is lyophilized and does not

contain stabilizers and fillers.
→ It looks empty.

 Store at 2～8℃

Components Amount

Endotoxin Indicator Vial (> 1,000EU/mL) 25 vials

Criteria for determining the effectiveness

of the dry heat sterilization process

Efficacy is judged from the

difference in endotoxin
Confirming the reduction concentration between
rate of endotoxin Unbaked and baked
concentration
Unbaked (Y/X) Baked
X EU/ml Y EU/ml

It is up to each user to determine whether it is effective in dry

heat sterilization (or other cleaning processes).
For reference, in the previous US Pharmacopoeia, it used to
consider effective when a reduction of at least 1/1000 (3-log
reduction) or more was confirmed.

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A flowchart of test method

 Which test method do you use?

Checking test method ・・・ ⇒ Gel-clot or Terbidimetric or Chromogenic method?
 Which LAL reagents and devices do you use?
→ Checking in advance
Preparation before dry
heat sterilization

Dry heat sterilization ・・・  Ex. 250℃ 30min

Dissolving ・・・  Dispensing endotoxin free water into each vials

Endotoxin concentration  Gel-Clot

measurement ・・・  Terbidimetric（Recommend）
 Chromogenic（Recommend） Unbaked Baked

Comparing the endotoxin concentration between

Analysis ・・・ unbaked vials and baked vials and confirming whether it
has been reduced by more than 1/1000 (3-log reduction).
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Checking test method

≪Recommendation≫
Selection of measurement method
→ All of method (Gel-clot, Turbidimetric, Chromogenic) are available. Turbidimetric
→ We recommend Turbidimetric or Chromogenic method. Chromogenic
Selection of LAL Reagents
→ All of LAL reagents are available.
ex. Turbidimetric method ES-F series
ex. Chromogenic method Color KY series

Selection of standard
→JP-RSE 、USP-RSE、CSE

Selection of device ≪Recommendation≫

→ET-7000、MPR etc
All of endotoxin measurement devices are available ET-7000

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Confirmation of installation location in dry heat

sterilizer (how many vials are necessary?)

Dry heat sterilizer

Ex.1） Ex.2）

→ 13 vials are needed →3 vials are needed

 Installation location (number of vials) → Determined by user
* There is no clear rule
* It’s better to check the point where the temperature seems to be
the lowest (if you know)
 Confirming the performance of dry heat sterilizer that you are using
【Source】 The handout provided by Dr. Honjo, Nippon Medical School Imaging Center for Healthcare 7

Dry heat dry heat sterilization

※ Preparing the same lot# of vials
≪Requirments≫
 Endotoxin Indicator Seal
 Dry heat sterilizer （Red prat）
 Endotoxin free water For bake For unbake
 Vortex mixier ○vials 2 vials
Rubber stoper

Label
Dry heat sterilization

Just place an appropriate number of Endotoxin indicator vials

(without removing seal, stopper or label) in a dry heat sterilizer.
Endotoxin
indicator vials
For bake

Dry heat sterlizer

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Dissolving
Dissolving
Seal

3 1. Removing seals and stoppers from baked vials

1 2 after returning to room temperature.
2. Removing seals and stoppers from Unbaked
Endotoxin vials
Free water Rubber
Each 1 ml
3. Dispensing 1 ml of endotoxin free water into
stopper
each vials
Baked Unbaked 4. Capping each vials not spill contents
5. Mixing for 30 min

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4 Mixing
※Repeating for each vials

※ Unbaked vials cannot be measured as they

are because the endotoxin concentration is high.
→ It needs to be diluted.
Baked Unbaked
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1 mL 1 mL

Diluting unbaked vials

Ex. ) In case, preparing a dilution series for standard curve of 0.05－5 EU/ml.

Unbaked
1 mL

2,000 EU/mｌ 0.05－5 EU/ml It can not be measured.

Ex. Diluting ※Unbaked vials should be diluted to be within the

X 1,000 detection sensitivity of 0.05－5 EU / ml.

Ex.）2 EU/mｌ 5 – 0.05 EU/ml

It can be measured

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Test method by Toxinometer®

Endotoxin concentration
measurement

Preparing a dilution series

for standard curve

Diluting unbaked vials

≪Requirments≫
 Endotoxin Indicaor
Endotoxin concentration →Baked vials ; (ex.）2 vials
measurement (Dry heat sterilizer ; one on the top, one on the bottome)
→Unbaked vials ：2 vials
 Device：Toxinometer ET-7000、PC
：Vortex mixer
 LAL reagents (ex.) ES-F Single test
Analysis （KTA Quantitative Range : 0.001-10EU/mL ）
 Standard：(ex.）CSE
 Endotoxin free water
 Test tube ：Dry heat sterilized tube
 Vials for dilution
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Test method by Toxinometer®

Preparing a dilution ※CSE：Control Standard Endotoxin
series for standard curve
Points for standard curve ：ex.）0.05, 0.5, 5 EU/ml

Diluting unbaked vials ≪Recommendation≫ Standard curve

Preparing a dilution series

Diluting unbaked vials to be within the
with 10-fold serial dilution.
detection sensitivity of 0.05－5 EU / ml.

Endotoxin concentration
measurement

↓Adding CSE dilution series, Unbaked vials (diluted), baked vials

to LALReagents （ES-F Single test）.
↓Endotoxin concentration measurement using Toxinometer®
Analysis
Comparing the endotoxin concentration between unbaked vials and baked vials and
confirming whether it has been reduced by more than 1/1000 (3-log reduction).

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Test method by Toxinometer®

Analysis <Calculation of the minimum log reduction>

Endotoxin concentration of unbaked vials（EU/ml） 1．Calculating endotoxin concentration baked vial（Y EU/ml),
and unbaked vial （X EU/ml）
Vial ① Vial ② Avarage
2． Assigning the endotoxin concentration of each vial to the
4000 4400 4200
following formula.
※If the endotoxin concentration of unbaked vial is under
the detection sensitivity (<0.050 EU / ml) of the standard
Endotoxin concentration of baked vials and Result curve (0.05-5 EU / ml), Calculate as Y = 0.050 EU / ml
Endotoxin
Installatio Log Log reduction = log(X)－log(Y)
concentrati Result = log(4200)－log(0.050)
n location on（EU/ml）
reduction
= 3.62－(－1.30)
1/16,700 = 4.92
Vial (A)-1 ＜0.050 (4.22 log)
Valid
⇒Reducing by more than 1/1000 (3-log reduction).
1/16,700
Vial (A)-2 ＜0.050 (4.22 log)
Valid
1/16,700
Vial (B)-1 ＜0.050 (4.22 log)
Valid
1/16,700
Vial (B)-2 ＜0.050 (4.22 log)
Valid

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Application consideration
 Each vials have variations in endotoxin concentration.
※ It’s recommended to prepare 2 vials of unbaked vials and calculate the average
value.

・This product guarantees > 1,000 EU/mL.

✓It’s recommended to measure once per year.

It’s recommended that the label display confirmation test be

performed in the measurement environment in which it will be
used first.

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