OBJECTIVE:

To determine the Ethylene oxide sterilization process consistently performs as intended by running
the system and recording all relevant information. The Data and Test results mist demonstrate that
the process meets pre-determined specifications and under normal conditions as well as worst case
conditions.

SCOPE:

The scope of validation protocol is to provide sterilization validation strategies for ethylene oxide
sterilization of medical device. This document will shoe two approaches for reducing or eliminating
bioburden on medical device. This will also show test method, sampling method and acceptance
criteria used in validating ethylene oxide sterilization of medical devices.

RESPONSIBILITY:

Person Responsibility
Validation team  Preparation of protocol
 Organizing validation activities
 Collecting the samples and sending to QC
 Review and interpretation of final results
 Preparation of reports
Quality control  Review of proto9col and report
 Analysing the test samples
 Reporting and interpretation of results
Production  Review of protocol and report
 Conducting the validation activity as per the protocol
Quality assurance manager Review and approve the validation protocol

STNADARDS FOR REGULATORY REQUIREMENTS:

 ISO 11135:2014, Sterilization of health-care products — Ethylene oxide — Requirements for

the development, validation and routine control of a sterilization process for medical devices
 ISO 10993-7, Biological evaluation of medical devices- Part 7: Ethylene oxide sterilization
residuals
 ISO 11737-1, Sterilization of health care products — Microbiological methods — Part 1:
Determination of a population of microorganisms on products
 ISO 11737-2, Sterilization of health care products — Microbiological methods — Part 2: Tests
of sterility performed in the definition, validation and maintenance of a sterilization process

LIST OF DOCUMENTS:

 SOP for ethylene oxide sterilization process

 SOP for environmental monitoring
 SOP for sampling procedure
 SOP for testing method
 SOP for material handling including biological indicator
 Sterilization cycle parameter specification sheet

PRE-REQUISITE:
The following requisite must be fulfilled before the ethylene oxide sterilization process validation.

 Equipment qualification: - Equipment associated with ethylene oxide sterilization process

must be qualified prior to process qualification.
 Calibration: - all process sensing, controlling, indicating, and recording devices on the
sterilizer or independent systems associated with the sterilizer must be calibrated and
recorded. Calibration program must be documented and detailed procedure of calibration
frequency for all the instruments should be identified.
 Product qualification: -
 Product and packaging material evaluation: - as per ISO 11135 product and
packaging material must be evaluated for ethylene oxide and humidity penetration.
 Product grouping: - For efficient and cost-effective performance validation similar
device can be grouped in to families. A family of products can be considered to be all
those products of similar design and material of construction, similar bioburden
levels but can be consist of different sizes.
Devices can be grouped based on several criteria:
 Similarity of materials of construction
 Same product design of different sizes, lengths or thickness
 Design complexity
 Similar method of manufacture
 Multiple combinations of devices
 Amount and type of packaging
 Selection of family representative: - each family of products will contain a number of
devices. From these devices, the representative challenge to the sterilization process and will
be the most difficult to sterilize device in the family group and will be sued as the BI carrier.
Following criteria can be used to select the family representative: -
 The highest bioburden
 Smallest opening into the interior of a device
 Must subassemblies
 Most convoluted passageways
 Most dense package configuration

Process and Validation Approach: -

 Parameters to be Verified

Bioburden Assessment No. of microorganism on product

Loading in chamber Loading Pattern

Chamber 1. Load temperature

pre-conditioning 2. Humidity of load
3. Duration

1. Initial evacuation / vacuum

Conditioning 2. Evacuation rate

3. Product load temperature

Gas injection and 1. Gas injection rate

dwell 2. Pressure rise upon admission of
Gas

3. Duration of Exposure

Post exposure gas  Vacuum rate

purge (Aeration)  Number of Air wash
Process Validation Procedure: -

 Bioburden assessment: - an understanding of the population of viable microorganisms on

the finished device (bioburden) is necessary and required to support the validation process.
The bioburden assessment includes the total number of microorganisms with their identities.
The identification needs confirmation of gram stain characteristics and genus. It provides
useful information and can be used to monitor changes over time and as a comparison to
organisms recovered during environmental monitoring. For determination of bioburden
Refer to Appendix 2.
 Selection of Process Challenge Device (PCD): - Process challenge device can be selected
from the family representatives in order to reduce the number of products tested during the
validation. Internal PCD is the most difficult to sterilize device seeded with a BI. For ease of
sample removal an external PCD can be used in the validation.
External PCDs are the external BI test pack that replaces the internal PCD. External PCD
should serves as a resistance greater than or equal to that of the internal PCD. The external
PCD is usually placed on the outside of the sterilization load between cases, just inside the
case or under the stretch wrap.

 BI or PCD placement in the product load: - after the product load challenge has been
identified, the BI or PCD positioning and placement can be determined. BIs and PCDs should
be distributed throughout the product load and, as much as possible, in the same orientation
(e.g., vertical).

Following minimum number of Bis/PCDs to be included in each validation cycle (as per ISO
11135): -

 Up to 10 m3 the number of Bis is m3, with a minimum of 5.

 From 10 m3 up to 100 m3, the number of additional Bis is one per additional cubic
meter.
 Temperature and Humidity sensor in product load: - for humidity check in preconditioning
and conditioning/sterilization one humidity sensor per 2.5 m3, with minimum number of
sensor is two should be placed in sterilization load and it should include pallet centers, edges
and surfaces.
for temperature monitoring in validation one temperature sensor per cubic meter of product
volume with a minimum of 3 sensors should be placed in the load.

Load Temperature: - ISO 11135:2014 requires that the minimum temperature of product
permitted to enter preconditioning be determined. Load temperature prior to entrance to
preconditioning should be determined at the lowest temperature zone. Direct placement of
cold loads into preconditioning can result in excessive water condensation and load wetting,
which can cause product damage and reduced lethality. In process validation it requires to
simulate the worst condition to which the load will ever be exposed. The anticipated load
temperature extremes can be simulated during validation by following techniques described
in the appendices 1.

Identification of the worst case location or cold spot: - temperature is the easiest variable to
measure and monitor, therefore temperature is used as an indicator of the worst case
location in the sterilization load. During the preconditioning or conditioning phase
 temperature profile against time of the sterilization load measured and based on that data
the worst case location or cold spot is identified.

Process Qualification Runs: -

For validation, a microbial challenge will be performed to demonstrate the adequacy of the
process to achieve the desired Sterility Assurance Level (SAL). Two methods are used
a) Half cycle method (Overkill approach)
b) Bioburden / BI method

a) Half Cycle Method: - during the bioburden assessment, if characterization of bioburden is

not performed and the bioburden level is more than 100 CFU than the following steps are
performed. In this method one fraction cycle, 3 half cycle and one full cycle is performed.

Place Biological Indicator (BI) with 106 spores of Bacillus atropheus in the PCDs and product
samples in each pallet with humidity and temperature sensor. (place PCDs to cold spot of the
product load).

Load pallets of sterilization load in to the sterilization chamber

Run fractional cycle according to prescribed cycle parameter

(use 1⁄4 or ⅙ gas exposure time than full cycle)

Aerate the product

Remove the product sample and PCDs from the load

Perform sterility test on product sample and Bis.

Additionally perform Bacteriostasis / Fungi statis test on the product sample.

If product sterility samples show survival, repeat the run on new load with elevated gas exposure
time until no survival in product and growth in Bis (evaluation of BI appropriateness)

Run 3 consecutive half cycle according to prescribed cycle parameter

(Use 1/2 gas exposure time than full cycle)
Aerate the load
 Remove the product sample and PCDs and perform sterility test on sample and BIs

Sterility test should show no growth for both product and BIs samples

Perform one full cycle (routine process exposure time) on the aerated sterilization load.

Place addition product sample for the additional test.

Aerate the load

Remove the product sample and PCDs and perform following test on product sample

1. LAL test (pyrogen test)

2. Product and packaging functionality test
3. Ethylene oxide residue test

b) Bioburden /BI method: - this method required that bioburden level be demonstrated to be
relatively consistent over time and the resistance of the bioburden be shown to be equal to,
or less resistant then, the resistance of the biological indicator. During the bioburden
assessment, if the characterization of bioburden performed and level of bioburden is less
than or equal to 100 CFU which indicating a lesser challenge than the BI than the following
steps can be followed.

Establish worst case location in the product load based on temperature distribution and
humidity of load

Place BI within known amount of microorganism in the PCDs, temperature and humidity sensor

Perform 5 fractional cycles with graded exposure time (e.g. 0, 4, 8, 12, 16 minutes)

Aerate the load and perform sterility test on BI

Calculate rate of inactivation (D value) using survival curve or fractional negative method

Calculate half cycle and full cycle exposure time

Place product samples and PCDs in each pallet with temperature and humidity sensor
 Run 3 half cycle according to prescribed cycle parameter

Aeration

Remove product samples and PCDs and perform sterility test on product sample and BIs. Additionally
perform fungi statis/Bacteriostasis test.

Perform one full cycle (routine process exposure time) on the aerated sterilization load. Place
addition product sample for the additional test.

Aerate the load

Remove the product sample and PCDs and perform following test on product sample

1. LAL test (pyrogen test)

2. Product and packaging functionality test
3. Ethylene oxide residue test

Maintenance of Validation: -

Requalification: -

1. Any significant change in product, manufacturing process, packaging, sterilization equipment

or process, product loading or density may necessitate requalification. A documented formal
review of any change should be undertaken to make this determination.
2. To ensure continued control, periodic repetition (annually is recommended) of all or part of
the performance validation should be considered.
Sampling method and test method: -
 Sampling method: -
Sample taken for validating sterilization process should be representative of entire batch and
it should be taken from most critical part of load configuration. Sample must be taken from
each separate packaging system place on.

LIST OF TESTS TO BE EXECUTED

No Test Objective References

1. Bioburden to check the liable ISO 11737:1 and ISO
determination microorganism in the product 11737:3
2. Sterility testing Product sterility testing is ISO 11737:2
determine any viable
microorganisms remain on
product during sterilization
3. Biological Indicator To challenge the sterilization ISO 11139
testing process and are used during
validation and routine
processing
4. Fungi statis / To determine the recovery ISO 11737:1
Bacteriostasis percentage, microbial growth
and will be used to correct
Bioburden results.
 5. LAL test To determine Endotoxin level TGO No. 50, USP
(pyrogen-end product of gram- (161) & (85)
negative bacteria)
6. Product package To evaluate the outer ISO 11607-1
functionality test packaging of a device.
7. Product functionality To evaluate the changes in the ISO 11607-1
test product characteristics.
8. Ethyle oxide residue To ensure the amount of ISO 10993-7
test ethylene oxide residual levels
are within limits

Test results and acceptance criteria: -

No. Test Acceptance criteria Test results
1. Sterility Testing No growth for product sample
2. Biological Indicator Fractional cycles: -
sterility testing
 No growth
Half cycle: -
 No growth
Full cycle: -
 No growth
All positive controls must show
growth
3. Fungi statis / Must be negative
Bacteriostasis
4. LAL test Endotoxin and pyrogen must be
absent
5. Product package Packaging integrity must be
functionality test intact
6. Product functionality Product should not show any
test degradation, discoloration and
physical changes
7. Ethylene Oxide residue Ethylene oxide residue levels
test must be in limit at the day of
release
8. Process parameters All parameter must meet with
specified cycle parameters

Process parameter check steps: -

Environmental preconditioning: -

Parameters to be checked Actual Specification

Temperature of
preconditioning room
Humidity of the
preconditioning room
Duration of preconditioning
 Acceptance Criteria met (yes / no)
Comments

Verified by: _________________ Date: _________________

Initial evaluation: -

Point of consideration Actual Specification

The evacuation rate (vacuum)
to maintain the seal integrity

Amount of negative pressure

Number of Nitrogen wash

Acceptance Criteria met (yes / no)

Comments

Verified by: _________________ Date: _________________

Humidification: -

Point of consideration Actual Specification

Level of moisture

Heat
 Acceptance Criteria met (yes / no)
Comments

Verified by: _________________ Date: _________________

Gas injection and gas dwell: -

Point of consideration Actual Specification

Gas concentration

Duration of exposure

Acceptance Criteria met (yes / no)

Comments

Verified by: _________________ Date: _________________

Post exposure gas purge and air in bleed: -

Point of consideration Actual Specification

Number of Nitrogen wash

Vacuum rate

Acceptance Criteria met (yes / no)

Comments

Verified by: _________________ Date: _________________

Heated aeration: -

Point of consideration Actual Specification

Temperature of room

Aeration duration

Acceptance Criteria met (yes / no)

Comments

Verified by: _________________ Date: _________________

Appendix 1 Simulation of Anticipated process Conditions

Anticipated load temperature extremes during transport, handling, and storage can be simulated by
following techniques: -

a) Use of cold storage – For example, the product load is stored under refrigeration or
deliberately induced cold temperature. The temperature of the storage area should be less
than or equal to the lowest temperature the product is expected to be exposed to
throughout the year. Product temperature data are recorded during the storage time. The
time in storage should be equal to or greater than the maximum time any product load is
expected to exist under such conditions, or the maximum product temperature while in
storage becomes the minimum acceptable product temperature for a load to be admitted to
the preconditioning phase. This applies to preconditioning or to conditioning when
preconditioning is not used. The use of refrigeration can result in unrealistically low humidity
levels, with the resultant desiccation of the load. A desiccated load can be much more
difficult to sterilize than a non-desiccated load.

b) Temperature modelling – data analysis of load temperature studies may be augmented by

modelling to establish the additional time required for starting temperatures lower than
those studied. If lower starting temperatures are allowed, the effect of additional
condensation should be evaluated. For example, at the time of validation, the lowest product
load temperature point is 60 ˚ F prior to entering preconditioning. Once in preconditioning,
data yield a temperature profile for the worst-case position showing that the load
temperature increases to 100 ˚ F by the end of preconditioning. When the temperature over
time data are graphed, the linear part of the time/temperature relationship (which is
generally the initial few hours of preconditioning when the difference between load
temperature and preconditioning area temperature is the greatest) can be used to
extrapolate the necessary preconditioning times for sterilization load at temperatures lower
 than 60 ˚ F. Using this technique to calculate a minimum preconditioning time will yield a
time that is greater than that required. Other techniques of temperature modelling can be
used.

c) Seasonal Validation – preconditioning validation can target those seasons that present the
most extreme temperature and humidity conditions. For example, a validation performed
during the coldest part of the year (which would also present the lowest ambient humidity
level) could yield minimum preconditioning parameters valid for routine production cycles
during the entire year. However, in cases of validations performed during the summer, it is
advisable to validate the precondition process with simulated cold storage, mathematical
modeling or to repeat the preconditioning validation at least one during the winter to
confirm the validity of the parameters.

Appendix 2: - Determination of Bioburden

Determination of Bioburden:
 collect ten (10) each product samples randomly from three (3) different batches of routine
production processes.
 Send to approved test lab for bioburden evaluation (enumeration of aerobes, fungus, and
spores.) at the test lab, perform the bioburden determinations using a standard method as
outlined in ISO 11737-1 by extracting each device individually and filtering the extract
through a sterile bacterial retentive filter.
 A validation of bioburden recovery should be performed. (an additional five non-sterile
samples are required).
 Determine the average bioburden per device for each lot, as well as the overall batch
average bioburden.
 Calculate the overall average bioburden. If a spike (a single value at least 2 X the overall
average) occurs, the spike value may be used rather than the average for the cycle selection.
Sample item portion (SIP) method for bioburden determination:
Based on following criteria product samples portion determined fir bioburden assessment.
1) If the product with an average Bioburden equal to or grater than 1.0 whenever practicable,
an entire product (SIP equal to 1.0) should be used for testing in accordance with ISO
11137:2. When the use of an entire product is not practicable, a selected portion of product
(sample item portion) may be substituted. The SIP should be as large a portion of item as
practicable in order to manipulate in the laboratory, and should be of a size that can be
handled during testing.
2) If a product with an average Bioburden equal to or less than 0.9, and entire product (SIP
equal to 1.0) shall be used for testing in accordance with ISO 11137:2.
3) If the bioburden is evenly distributed on and/or in the item, the SIP may be selected from
any portion of the item. If the bioburden is not evenly distributed, the SIP shall consist of
portions of product selected at random, which proportionally represent each of the
materials from which the product is made. If the bioburden distribution is known, the SIP
may be selected from the portion of the product that is considered to be the most severe
challenge to the sterilization process.
The value of SIP can be calculated based on length, mass, volume or surface area
Basis for SIP Product
Length Tubing (consistent diameter)
 Mass Powders
Gowns
Implants (absorbable)

Volume Fluid

Surface area Implants (non-absorbable)

Appendix 3 (GLOSSARY)

ETHYLENE OXIDE STERILIZATION GLOSSARY

Below is an alphabetical list of terms that may be used in discussions of ethylene oxide (EO)
sterilization as recognized by medical device manufacturers inspected by US FDA. All definitions or
explanations are to be taken as applied specifically in the context of EO sterilization processing.

Aeration
Part of the sterilization process during which ethylene oxide and/ or its reaction products desorb
(outgas) from the medical device until predetermined levels are reached. This may be performed
within the sterilizer and/or in a separate chamber or room.
Aeration area
Either a chamber or a room in which aeration occurs. The temperature in the aeration area is
elevated and controlled to assure that the aeration process is accelerated and repeatable. Air is
recirculated in the room to maintain food heat distribution with a portion of the air stripped off and
cleansed with the pollution control equipment. The stripping of the air reduces the amount of free
ethylene oxide resident in the rooms, thus prevents recontamination of product load.
Bacteriostasis/Fungi stasis Test
Test performed to evaluate the presence of microbial growth inhibiting properties of a healthcare
product. This test assures that any reaction of the growth medium with the different materials used
in the manufacture of the device will not mask or prevent growth from any viable organism
remaining on the device after being subjected to a sterilization process.
Bioburden
Population of viable microorganisms on a raw material, component, finished product, and/or
package. Unlike the gamma irradiation process, bioburden data is not utilized in ethylene oxide
sterilization to determine the sterilization process. It is collected to quantify the amount of product
contamination, which is then compared to the population of the biological indicator used in the EO
sterilization process.
Biological Indicator
Inoculated carrier contained within its primary pack ready for use providing a defined resistance to
the specified sterilization process.
Calibration
Comparison of a measurement system or device of unknown accuracy to a measurement system or
device of unknown accuracy (traceable to national standards) to detect, correlate, report, or
eliminate by adjustment, any variation from the required performance limits of the unverified
measurement system or device. All critical measuring devices utilized on ethylene oxide sterilizers
which may impact the quality of the process are calibrated to traceable national or international
standards.

Chamber
Enclosed area which only accommodates sufficient product to fill the sterilizer. EO chambers are
constructed of steel or stainless steel and are designed to withstand the extreme pressures and
elevated temperatures utilized in the EO sterilization process.

Commissioning
Obtaining and documenting evidence that equipment has been provided and installed in accordance
with its specification and that it functions within predetermined limits when operated in accordance
with operational instructions. All EO sterilization equipment which complies with ANSI/AAMI/ISO
11135 is commissioned.
Conditioning
Treatment of product within the sterilization cycle, but prior to sterilant admission, to attain a
predetermined temperature and relative humidity. This part of the sterilization process may be
carried out either at atmospheric pressure or under vacuum (see also preconditioning).
Critical parameters
Parameters identified as being essential to the sterilization process and requiring monitoring.
D-Value
Time (expressed in minutes) required to achieve inactivation of 90 percent of a population of a test
organism under stated exposure conditions. Also referenced as the D10 Value or decimal reduction
value.

Exposure Time
Time for which the sterilizer chamber is maintained within the specified range for temperature,
sterilant concentration, pressure, and relative humidity. Also, may be referred to as the time for
which a medical device (load) is exposed at the specified sterilization conditions.

Failure
Event in which a component does not perform one or more of its required functions within the
specified limits under specified conditions.

Flushing
Procedure by which sterilant is removed from the load and chamber by either multiple alternate
admissions of filtered air or inert gas and evacuations of the chamber or continuous passage of
filtered air or inert gas through the load and chamber.
Inactivation
Loss of the ability of microorganisms to grow and/or multiply under specified culture conditions.
Indicator
Combination of indicator agent and its substrate in the form in which it is intended to be used.
Inoculated Carrier
Carrier on which a defined number of test organisms have been deposited.
Installation Qualification
Obtaining and documenting evidence that equipment has been provided and installed in accordance
with its specifications and that it functions within predetermined limits when operated in accordance
with the operational instructions.
Manufacturer
Natural or legal person packaging or sterilizing a medical device.
Medical Device
Any instrument, apparatus, appliance, material or other article whether used alone in combination,
including the software necessary for its proper application intended by the manufacturer to be used
for human beings for the purpose of:
 Diagnosis, prevention, monitoring, treatment, or alleviation of disease;
 Diagnosis, monitoring, treatment, alleviation of, or compensation for injury or handicap;
 Investigation, replacement, or modification of the anatomy or of a physiological process;
 Control of conception; and which does not achieve its principal intended action in or on the
human body by pharmacological, immunological, or metabolic means, but which may be
assisted in its function by such means.
Microbial Barrier
Ability of the packaging system to prevent the ingress of microorganisms under specified conditions.
Microbiological Challenge
Biological indicators, biological-indicator test packs, or inoculated product that contain known
populations of microorganisms and can be used in testing sterilization cycles.
Overkill Sterilization Process
Process which is sufficient to provide at least a 12-logarthimic reduction or 12 D inactivation of an
appropriately resistant biological indicator with an established D value.
Package Integrity
Unimpaired physical condition of a final package.
Parametric Release
Declaring product as sterile based on physical and/or chemical process data rather than on the basis
of sample testing or biological indicator results.
Performance Qualification
Obtaining and documenting evidence that the equipment, as commissioned, will produce acceptable
product when operated according to the processing specifications.
Positive Sterility Test
Sterility test samples which exhibit detectable microbial growth after incubation.
Preconditioning
Treatment of product prior to the sterilization cycle in a room or chamber to attain specified limits
for temperature and relative humidity.
Preconditioning Area
Either a chamber or a room in which preconditioning occurs.
Process lethality
Capability of the sterilization process to destroy microorganisms.
Process Qualification
Obtaining and documenting evidence that the sterilization process will produce acceptable health
care products.
Product
Generic term used to describe raw materials, intermediate products, subassemblies, and finished
medical devices.
Product Qualification
Obtaining and documenting evidence that the health care product will be acceptable for its intended
use after exposure to the sterilization process.
Qualification
Documented evidence that all prescribed design and performance requirements are met.
Requalification
Repetition of part of all of the validation test requirements for the purpose of reconfirming process
reliability.
Sterilant
Microbicidal agent in the physical form in which it is active.
Sterile
Free from viable microorganism.in practice, no such absolute statement regarding the absence of
microorganisms can be proven (see sterilization).
Sterility
State of being free from viable microorganisms. In practice, no such absolute statement regarding
the absence of microorganisms can be proven (see sterilization).
Sterility Assurance Level (SAL)
Probability of a viable microorganisms being present on a product unit after sterilization. SAL is
normally expressed at 10-n.
Sterility Test
Test performed to determine if viable microorganisms are present.
Sterilization
Validated process used to render a product free from viable microorganisms. In a sterilization
process, the nature of microbial death is described by an exponential function. Therefore, the
presence of viable microorganisms on any individual item can be expressed in terms of probability.
While this probability may be reduced to a very low number, it can never be reduced to zero. The
probability can be expressed as a sterility assurance level (SAL).
Sterilization Cycle
Defined sequence of operational steps designed to achieve sterilization that is carried out in a sealed
chamber. Specifically, for EO sterilization, the treatment in a sealed chamber comprising air removal,
conditioning, injection of sterilant, exposure to ethylene oxide, and removal of ethylene oxide.
Sterilization Load
Goods that are to be or have been sterilized simultaneously in the same sterilization chamber.
Sterilization Process
All treatments which are required to accomplish sterilization, including preconditioning, the
sterilization cycle, and aeration.
Validation
A documented procedure for obtaining, recording, and interpreting the results needed to show that
a process will consistently yield a product complying with predetermined specifications. Validation is
considered as a total process that includes written protocol, evidence that the equipment as installed
meets design criteria and specifications (equipment qualification), use of calibrated instruments to
collect data, and evidence that the equipment can deliver the process within the specified tolerances
under established operating conditions and is reproducible as demonstrated by replicate runs and
process challenges (performance qualification).