PM-970-1

April 12th, 2015

Concise report of validation of sterilization according to ISO 11135:2014

1. General

1.1 The last revalidation was performed during the year 2001, and the previous
one that included revalidation of complete 3 cycles, was performed during
1996. Since then, at least once a year, requalification of the process is
performed according to ISO 11135. At this time, a review of the validation
system is performed, to see if there were any changes that require a new
validation.
The review contains:
1.1.1 Are there any changes in the chamber?
1.1.2 Are there any changes in the procedure?
1.1.3 Are there any changes in the line of products?
1.1.4 Are there any changes in the formula of the Gas?
1.1.5 Since the last checkup, were there more than 3 consecutive failures?
1.1.6 When reviewing the Sterilization Reports, is there any sign indicating that a
new validation has to be performed?
The Q.C. system performs such a review every year, and since the last
validation, no reviewed validation was required.

1.2 ISO 11135:2014 requires change in the quantities of temperature and humidity
sensors and the change of biological indicators compared to former ISOs. It
has to be stated that as for the routine control, we are using 24 biological
indicators in each sterilization process.
All products, manufactured and sterilized by Eurobands ETO sterilization are of
the same Product family (3.33).
1.3 Procedure of sterilization: (All the sterilization procedure is performed in the
same cabin in one continuous procedure without intervals. Preconditioning, the
sterilization cycle, washes and aeration.)
Packs of the same product family (3.33) are placed in a sealed gas autoclave
cabin, and acclimatized to a temperature of 30oC (temperature peaks of +80 -20
are permitted), with moisture of 35% - 65% relative humidity.
After remaining under these conditions for 18 hours, the temperature is raised
to 32oC (temperature peaks of +80 -20 are permitted) for a further 2 hours.
Slow vacuum is created until a vacuum of 0.280 Bar is achieved.
Additional moisture to achieve 35% - 65% is inserted (Due to increase of
temperature). Vacuum is maintained in the cabin for 90 minutes.
 -2-

Ethylene Oxide gas (20% ETO+ 80% CO2) is inserted by pressure, until
pressure exceeds 1.44 Bar. Contact remains for 18 hours.
After full contact of 18 hours, at a temperature of 32oC (temperature peaks of
+80 -20 are permitted), and at pressure of 1.44 Bar, a vacuum is created by
removing the Ethylene Oxide until a vacuum of 0.280 Bar is reached.
Filtered air is inserted into the autoclave until atmospheric pressure is
reached. Vacuum is again applied, and after inserting filtered air once
again, the procedure is repeated 7 times consecutively. For ventilation, 1
additional smaller cycle, to 0.730 Bar, is performed to remove residues
of ETO before opening of the chamber.
Validation of each particular sterilization process Conformity Tests:
During the process of loading the autoclave, as routine control, 24 packs of pre
designed tubes containing nX106 different bacteria and spores are placed in 24
positions in the autoclave, according to pre determined positions. These
spores are tested for efficiency of sterilization. Results are obtained after 48
hours.
12 other packs, without spores and bacteria are placed in pre determined and
fixed positions and again tested for sterilization efficiency of gram positive and
gram negative bacteria. Results are obtained after 14 days.
In addition to microbial tests, 24 chemical detectors, as part of the biological
indicator that are marked on it to show efficiency of the gas.
Minimum 4 times per year, the residues of ETO in the products after 48 hours,
are tested according to ISO 10993-7:2008 with the highest limit of 10 PPM (10
gr. Per litter).

2. Preparation for Validation process:

2. 1 Introduction of 25 temperature sensors and 10 humidity sensors after being
pre-validated for the accuracy of 0.50C of the temperature and 1% of relative
humidity, into the chamber plus 1 sensor of temperature and one sensor
humidity kept out of the chamber. The 35 sensors are introduced into the
chamber through a 38mm opening in the side of the chamber, - see attached
photograph.
2. 2 The pressure gage of the chamber was validated on July 2012 during the
annual validation of measuring devices, this routine procedure is performed
annually and the last validation was performed during 2014.
2. 3 Chamber loaded with 6 pallets containers that contain products for sterilization.
Each container is approximately 220cm wide, 180cm deep and 180cm height
and the distance between each pallet is minimum 5cm to permit air ventilation.
2. 4 The 25 pre validated temperature sensors - numbered from 1 25 are placed
into the packs in the pallets as per attached diagram.
2. 5 The 10 pre validated humidity sensors numbered from 1- 10 are placed into the
packs in the pallets as per attached diagram labeled in diagram no. 2.
Temperature pre validated sensor no. 26 and pre validated humidity sensor no.
11 are placed outside the pallets in the chamber near the center of the
chamber, at a distance of about 40 cm from the ceiling.
2. 6 The temperature sensor of the sterilizer was placed between carriage no. 3 and
4, at a distance of 90 cm. from the base of the carriages.
2. 7 Measurements: The total volume of the chamber is 24M3 and the goods are
placed on 6 pallets. Distance between the pallets and the side walls of the
 -3-

sterilization walls ~ 8 cm, Distance between the top layers of the pallets to the
ceiling ~ 10 cm. Distance between each pallet minimum 5 cm.
2. 8 Each pallet was covered with polyethylene sheet and wrapped with shrink
polyethylene.
2. 9 44 biological indicators of 1.6X106 were placed into 44 rigid packs of PVC sealed
by Tyvek lid. The packs were placed into the pallets according to the attached
diagram. One biological indicator from same lot was placed outside the
chamber.
2. 10 24 packs containing actual products simulating the contents of the sterilized
products were placed in pairs according to enclosed diagram labeled with
numbers from 1-12 and 1a-12a for each pair.
2. 11 Samples of packaging packs were tested for peel strength before sterilization
(QA-0707) and the data was recorded. The same lot of samples were marked
and placed in the chamber for sterilization.

3. Process and results

3. 1 Sterilization Lot number 882-12-12 started on December 18th at 13:30 and
ended the sterilization process at December 19th 18:30 and afterwards 7
washes and 1 ventilation (aeration) was performed all as explained in Par # 1.3
above.
3. 2 After 5 hours from the begin of process the minimum temperature of 300C to
320C was achieved in all 24 temperature sensors. The temperature stabilized at
temperature of 300C to 300C for rest time of acclimatization. The 10 humidity
sensors showed in the beginning an average of 45% and went down to 31%
average within an hour. At this stage steam was inserted to the cabin and the
humidity raised to 54% immediately. After 18 hours of acclimatization, the
temperature was raised on all 24 sensors within 1 hour and reached the
temperature of 320C to 340C and stabled at 320C for 1 additional hour, while
increasing the temperature, the average humidity dropped to 48% average,
steam was released into the cabin and the humidity raised to 54% and later to
61% in which it stabilized during all the process of contact time of the gas.
During this time the inner pressure was reduced to 0.280 bar. At 08:00 on
December 18th, ETO gas started entering the chamber until the pressure of
1.44 bar was achieved at 11:30. Contact time of the gas was set for 6 hours,
instead of the regular 18 hours of contact time. ISO 11135 requires half time
process, but as we did in the past, we have performed 1/3 of the regular
contact time. During contact time, the temperature increased in some sensors
to 340C but within 1 hour stabilized at 320C. The humidity was between 60% to
62%. 7 washes started after 6 hours of contact and the goods were released
from the cabin the next morning at 06:30.
3. 3 The results and the data in this revalidation of sterilization, did not differ from
the previous validations performed during 1996 and a revalidation performed
during 2001.
3. 4 Out side temperature during the process can be seen in Chart 3.
Out side humidity can be seen in chart 4.
3. 5 After the goods were taking out, the 44 biological indicators that were in the
chamber during the process turned into dark brown at the line that shows the
efficiency of the ETO, and 1 biological indicator that was not in the chamber, all
of them from the same lot, were placed into the incubator at the temperature
of 350C for 48 hours. After 48 hours, the 44 biological indicators that were in
 -4-

the chamber stayed green color as before incubation, the biological indicator
that was not in the chamber was yellow after first inspection of 24 hours.
3. 6 12 simulated products out of the 24, were incubated for 14 days in medias for
gram positive and gram negative bacteria. After 14 days, all simulated products
showed that they are sterile.
3. 7 Packs that were from the same lot of peel strength, were tested for peel
strength after sterilization. All packs were in the limit of QA-0707, and there
were no significant difference in the peel strength compared to those tested
before the sterilization process.

4. Conclusion:

4.1 The revalidation did not differ from the former validations conducted during
1996 and the revalidation conducted during 2001. In all former validation and
this revalidation (only 20 temperature sensors were used), the temperature
reached the 300C during the first 4 hours, and stayed stable till the temperature
was raised to 320C after total acclimatization of 18 hours. During sterilization
the temperature was stable at 320C. As 1/3 of contact time was used, far less
than required in ISO 11135 for half time, and all biological tests showed that
the 44 biological indicators were found sterile, the validation is approved
according to ISO 11135:2014.

5. Second biological revalidation

5.1. On January 13th, 2013, another revalidation was performed, in sterilization lot
883-01-13.
5.2. Samples of packaging packs were tested for peel strength before sterilization
(QA-0707) and the data was recorded. The same lot of samples were marked
and placed in the chamber for sterilization.
5.3. 44 biological indicators of 1.6X106 were placed into 44 rigid packs of PVC sealed
by Tyvek lid. The packs were placed into the pallets according to the attached
diagram. One biological indicator from same lot was placed outside the
chamber.
5.4. 24 packs containing actual products simulating the contents of the sterilized
products were placed in pairs according to enclosed diagram labeled with
numbers from 1-12 and 1a-12a for each pair.
5.5. 6 hours of ETO contact time were used in this revalidation instead of 18 hours
during regular sterilization. 1/3 instead of half time as requested by ISO 11135.
5.6. The process ended on January 16th 2013, and the chamber was emptied at
6:30 in the same morning.
5.7. The 44 biological indicators that were inside the chamber plus the 1 that was
outside the chamber were incubated for 48 hours at the temperature of 350C.
The 44 biological indicators that were in the chamber, remained green after 48
hours of incubation, the biological indicator that was not in the chamber was
yellow after being looked at during the next day within 24 hours from start of
incubation.
5.8. 12 simulated products out of the 24, were incubated for 14 days in medias for
gram positive and gram negative bacteria. After 14 days, that ended on
January 31st, 2013, all simulated products showed that they are sterile.
5.9. Packs that were from the same lot of peel strength were tested for peel
strength after sterilization. All packs were in the limit of QA-0707.
 -5-

Conclusion of the second revalidation of lot 883-01-13.

5.10 As 1/3 of contact time was used, far less than required in ISO 11135 for half
time, and all biological tests showed that the 44 biological indicators were
found sterile, and the 12 simulated products we found to be sterile after 14
days of incubation that ended on January 31st 2013, the validation is approved
according to ISO 11135:2014.

Third biological revalidation

6.1. On February 3rd, 2013, another revalidation was performed, in sterilization lot
884-02-13.
6.2. Samples of packaging packs were tested for peel strength before sterilization
(QA-0707) and the data was recorded. The same lot of samples were marked
and placed in the chamber for sterilization.
6.3. 44 biological indicators of 1.6X106 were placed into 44 rigid packs of PVC sealed
by Tyvek lid. The packs were placed into the pallets according to the attached
diagram. One biological indicator fro same lot was placed outside the chamber.
6.4. 24 packs containing actual products simulating the contents of the sterilized
products were placed in pairs according to enclosed diagram labeled with
numbers from 1-12 and 1a-12a for each pair.
6.5. 6 hours of ETO contact time were used in this revalidation instead of 18 hours
during regular sterilization. 1/3 instead of half time as requested by ISO 11135.
6.6. The process ended on February 5th 2013, and the chamber was emptied at
9:00 in the same morning.
6.7. The 44 biological indicators that were inside the chamber plus the 1 that was
outside the chamber were incubated for 48 hours at the temperature of 350C.
The 44 biological indicators that were in the chamber, remained green after 48
hours of incubation, the biological indicator that was not in the chamber was
yellow after being looked at during the next day within 24 hours from start of
incubation.
6.8. 12 simulated products out of the 24 were incubated for 14 days in medias for
gram positive and gram negative bacteria. After 14 days, that ended on
February 19th, 2013, all simulated products showed that they are sterile.
6.9. Packs that were from the same lot of peel strength were tested for peel
strength after sterilization. All packs were in the limit of QA-0707.

Conclusion of the third revalidation of lot 884-02-13.

6.10 As 1/3 of contact time was used, far less than required in ISO 11135 for half
time, and all biological tests showed that the 44 biological indicators were
found sterile, and the 12 simulated products we found to be sterile after 14
days of incubation that ended on February 21st 2013, the validation is
approved according to ISO 11135:2014.

ETO residues:
ETO residues to be tested on regular sterilization cycle, of 18 hours contact
time, of Lot 885-02-13 were sent for finding the ETO residues according to
ISO 10993:2009. 10993-7 2008-10-15 Ethylene oxide sterilization residuals.
In any event, we are performing 4 ETO residues per year.

Summarized by Michael Porat from the data in the validation process file.
April 12th, 2015
 STERILIZATION AREA ON ROOF
13

E B F
2
7 5
11 A D
4
10 3
8
C
1
G
6 9

12

Read:
1. Sterilization unit ETO: Inside sizes; length 730cm X height 230cm X width 220cm.
Inside room for pallets; length 720cm X height 210cm X width 180cm.
2. Scrubber for ETO gas absorption. Total capacity 23 mt3. Actual use 1.63 mt3.
3. Vacuum pump 25 HP, water cooling sys.
4. Steam generator gas operated.
5. Water jar for steam generator.
6. Computer operator in separate room.
7. ETO cartridges 20% ETO/80% CO2.
8. ETO residues detector. (connected to a ringing bell).
9. Compressed air cartridges for backup.
10. Compressed air generator starts when pressure is less than 6 Bar stops 8 Bar.
11. Compressed air generator starts when pressure is less than 4 Bar stops 8 Bar
12. Concrete wall.
13. Wire fence.

A. Empting tube from 1,400 millibar to atmospheric pressure.

B. Tube for empting AIR/ETO from sterilization unit to vacuum pump.
C. Tube for water from scrubber to cool vacuum pump.
D. Tube for water from vacuum pump to scrubber.
E. Chimney from scrubber to atmosphere , 8 meter over roof.
F. Steam from generator to heat ETO gas and heating of inner sterilization unit.
G. Soft water for steam generator.