EO Sterilization: The BasicsPresenter: Lisa Foster

Lisa.foster@medpoint.com

Topics

• How does EO sterilize• Basic factors affecting sterilization• Calculating D value• Biological Indicators• An EO Cycle• Cycle Validation• EO Residues• Product Release

Governing Standard• ANSI/AAMI/ISO 11135: Medical Devices –

Validation and routine control of ethylene oxide sterilization.

• www.aami.org

How Does EO Sterilize?

EO as a Sterilant

EO is the simplest epoxy• Boiling point 10.4°C (50.7°F)• Extremely reactive• Kills by alkylation

Irreversibly binds to key molecules in cellDNAProteins (enzymes)

EO as a Sterilant

Molecules no longer function Cell no longer reproduces Cell dies

Basic Factors Affecting EO Sterilization

Four Basic Factors to Microbial Kill

•EO concentration•Water Vapor (Humidity)•Temperature•Time

EO Concentration

•Commonly 400 and 700 mg/l •As EO concentration increases at a

given temp and RH, ▫microbial inactivation (kill) rate

increases

EO Concentration

•Killing concentration required at the site where the microbes (BI’s) are located

Water Vapor (Humidity)

•Required for EO to react with the critical cell molecules

Generally measured as Relative Humidity

Temperature

•Kill rate increases with temperature▫D-value decreases (time) with

temperature▫Exponential function

•For each 10°C (18°F) rise in temperature, the spore inactivation rate will generally double

Time

•Amount of kill increases with exposure (EO gas dwell) time

•90 percent of surviving microbes are killed for each D-value time in gas dwell

D-Value

•Determined to prove predictable logarithmic death kinetics of challenge microorganisms (BIs) or natural bioburden

D-Value

•“Decimal” Reduction Time - Measure of the biological organism’s resistance to the sterilant

•Time in minutes necessary to reduce (kill) a microbial population by one logarithm or 90%

A Simple D-Value Calculation

TimeLog of Starting Population – Log of Final

Population

Example•1,000,000 or 106 spores. (the Log = 6)•20 minute gas dwell•Result is 10,000 or 104 spores

(the Log = 4)

Time = 20 minutesLog of Starting Population (which equals 6) – Log of Final Population

(which equals 4)

20 minutes6 – 4

Thus: D-Value is 20 divided by 2 = 10 minutes

What does that mean?

•It takes 10 minutes to go from:▫1,000,000 to 100,000▫ or 10,000 to 1,000▫ or 10 to 1▫ Etc.

Biological Indicators

Biological Indicators• In EO sterilization,

we use a paper strip impregnated with about a million spores . The strip is in a gas-permeable, bacteria-impermeable glassine envelope

Biological Indicators (BIs)

•More difficult to kill than natural occurring bioburden (microrganisms in or on product)

Biological Indicators (BIs)

•Traditionally, >106 spores of an organism (i.e., Bacillus atrophaeus) highly resistant to EO process

Spores

Bacillus???

•The name of the bacterium is commonly Bacillus subtilis or B. subtilis.

• It has been renamed and is officially B. atrophaeus.

Biological Indicators (BIs)

•Provided in various forms for industrial applications:▫Placed in product to represent

most resistant location in product

Examples of Biological Indicators

SCBI Disc Strip SCBI

Placing the BI: The BI is positioned in the

Most Difficult Location for gas to reach

Testing EO-exposed BIs

•The BI strip or disc is planted into a test tube containing Tryptic Soy Broth (TSB).

•The strips are incubated at 30-35oC for seven days (USP).

A Positive

Orange pellicle

Cloudy

Sediment

TSA Agar(orange)

Tyrosine Agar (black)

The EO Cycle

Conventional Cycle•Three steps▫Preconditioning▫Sterilization ▫Aeration

Preconditioning•First “processing” step of EO

sterilization.•Ensures the load is always

processed at roughly the same temperature and humidity upon entry to the chamber.

Preconditioning

•Heat and Humidity Introduced to Load

•Air circulated to assist heat/humidity transfer

•Generally 18-24 hrs

Chamber

EO Sterilization Cycle

Generic 100 Percent Cycle

0.0

5.0

10.0

15.0

20.0

25.0

30.0

0 100 200 300 400 500 600 700

Time from Cycle Start (minutes)

Pres

sure

(inH

gA)

Initial Vacuum

Leak Test

Humidification

Humidity Dwell

Gas Inbleed

Gas Dwell

After Vacuum

Gas Wash

Final Release

Cycle Validation

•Rigorous microbiological and physical testing

•Beyond that used in routine monitoring for either:• Conventional• Parametric

Cycle Validation

• Conventional Cycle Run 3 Half Cycles with parameters established during Cycle Development Culture BIs (2 to 7 days)

Run minimum 1 Full Cycle to evaluate functionality and residuals

Cycle Validation

•Conventional Cycle (continued)▫Aeration Requirements

Develop dissipation curve to establish release time

Aerate long enough to assure safe residuals typically around 2 days on average

Cycle Validation

Conventional Cycle (continued)• Residual allowable limits are based on

application of product ISO 10993-7 Qualify release time based on three (3) separate lots.• May be satisfied with first two routine runs with end-point residual testing (to supplement dissipation curve)

Cycle Validation

Parametric ReleaseRelease on parameters, not BIAdded parameters; Load temp prior to Precon Load temp at end of Precon Chamber RH during humidity Chamber EO during dwell Load temp during dwell

Cycle Validation

Parametric ReleaseBI Requirements NONE

Cycle Validation

Critical step in overall validation • Periodic (typically annual)

requalification studies designed to detect inadvertant process changes

• Recommended upon introduction of new or significant changes in: Product Process Packaging Equipment

Revalidation

•Options:

▫Perform one (1) micro PQ run in each qualified chamber

Revalidation

•Options

▫Paper Revalidation: Documented evaluation of process to verify no process shifts throughout year. May be used as a tool, however not

recommended for continuous replacement of physical requalification.

Revalidation

AERATION

Aeration (Degassing)

•Last “processing” step.▫Aeration Room physically similar to

Preconditioning, but with no “Humidity”•Two methods of Aeration available▫Heated▫Ambient

Aeration (Degassing)

Heated Aeration•Heat Introduced to Load▫assist in degassing process

•Typically 2 days on average for Heated Aeration

Aeration (Degassing)

Ambient Aeration•generally has much longer minimum

time requirements

EO Sterilant Residues

EO Sterilant Residues

•What is a sterilant residue?•Why do we need to test?•Who tells us we have to test?•How do we test for these residues?

What is a Sterilant Residue?

Sterilant residues are compounds that remain on product after ethylene oxide sterilization.

Residues - What are They?

•Ethylene Oxide

•Ethylene Chlorohydrin - Formed when EO reacts with chlorine or chloride ions. Chlorine may come from salt or bleach.

•Ethylene Glycol - Formed when EO reacts with water.

Why Test?

•Patient safety.

•Regulatory limits.

Who Requires Testing?

•FR, Volume 43, No. 122- June 23, 1978 (FDA)

•ANSI/AAMI/ISO 10993-7

Factors Affecting Levels of EO

Residual amount depends on:▫Product materials▫Chamber gas concentration▫Gas dwell time▫Aeration conditions and length of aeration time

Product Release

Product Release

•Before or after aeration BI’s are pulled from load and sent to lab for testing (7 day test)

• If Parametric Release has been validated, load can be released immediately after aeration. (No BI’s required)

EO Sterilization: The BasicsPresenter: Lisa Foster

Lisa.foster@medpoint.com