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Residual Ethylene Oxide in Medical Devices and Device Material
Anne D. Lucas, Katharine Merritt, Victoria M. Hitchins, Terry O. Woods, Scott G. McNamee, Dan B. Lyle,
Stanley A. Brown
Center for Devices and Radiological Health, Office of Science and Technology, U.S. Food and Drug Administration
Received 6 November 2002; accepted 8 January 2003
Abstract: Ethylene oxide (EO) gas is commonly used to sterilize medical devices. The
amount of residual EO remaining in a device depends partly on the type and size of polymeric
material. A major concern is the amount of residue that may be available in the body. With
the use of the method described by AAMI for headspace analysis of EO residues, different
polymers and medical devices subjected to different numbers of sterilization cycles were
examined. Next, the effect of various extraction conditions and extraction solutions on these
polymers and medical devices was evaluated. The results showed different polymers desorb
EO differently. One polyurethane (PU 75D) had much higher EO residue than a different
polyurethane (PU 80A). Repeated extraction of the PU 75D was necessary to quantify total EO
residue levels. Different extraction solutions influence the amount and reproducibility of EO
detected, whereas multiple resterilizations showed no difference in amount of residual EO.
Bioavailability of EO was estimated by extracting the devices and polymers in water. Com-
parison of total EO residues to EO that was bioavailable showed no difference for some
polymers and devices, while others had an almost eightfold difference. Some standard bio-
compatibility tests were run on extracts and devices, but no significant effects were observed.
© 2003 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 66B: 548 –552, 2003
Keywords: ethylene oxide; medial device; polymer; bioavailable; toxicity
INTRODUCTION
About 20 –30% of the hospitals in the United States were
formerly reprocessing medical devices.
1
Now there is regu-
latory involvement on the reuse of medical devices, and many
hospitals have left the reprocessing of devices to contractors.
Still, this practice raises many issues, including possible
deleterious effects on the material properties, change in de-
vice performance, and risk of infection. The type of steriliza-
tion method used in reprocessing may have a significant
impact on the performance and safety of a reprocessed and
resterilized device. In the health-care center, one of the more
common ways to sterilize devices is to use ethylene oxide
gas. Ethylene oxide (EO) is used to sterilize medical devices
that cannot be sterilized by heat or radiation. EO sterilization
is relatively inexpensive,
1
but EO and some of its degradation
products are carcinogenic and mutagenic.
2– 4
One question regarding the impact of EO on resterilized
medical devices is the amount of EO residues remaining in a
device following repeated EO resterilizations. EO has been
reported to accumulate in some materials
5
. There are three
residue levels based on exposure categories: limited (daily),
prolonged (monthly), and permanent. Current allowable lim-
its for EO exposure from medical devices are 250 ppm
(recommended level from AAMI TIR 19 as a substitute for
testing for irritation and sensitization), with a daily maximum
human exposure of 20 mg EO for the first day, and 2 mg per
device for frequently used devices.
6,7
These standards have
sections on exhaustive extraction and simulated-use extrac-
tion for EO residues. The standards recommend simulated-
use extraction as the method of choice; however, this is
impractical for devices used for long periods of time, for
example, implants, for which exhaustive extraction is the
usual choice.
To address some of these issues, a series of studies were
conducted to examine residual EO levels in medical devices
and polymers with the use of the method developed by
ANSI/AAMI/ISO
7
for headspace analysis of EO residues.
The effects of various extraction conditions, extraction solu-
tions, and the number of resterilization cycles on the amount
of EO residues were evaluated. In addition, the total amount
of EO, using exhaustive extraction, was compared to the
amount EO that was bioavailable.
8,9
For this study bioavail-
able is defined as the amount of EO that may be assimilated
by the body; this is estimated by extraction of the material or
The opinions or assertions identified by brand name or otherwise are the private
views of the authors and are not to be construed as conveying either an official
endorsement or criticism by the U.S. Department of Health and Human Services or the
Food and Drug Administration.
Correspondence to: Anne D. Lucas, US FDA, CDRH/OST, HFZ 112, 12709
Twinbrook Pkwy, Rockville, MD 20852 (e-mail: adl@cdrh.fda.gov)
© 2003 Wiley Periodicals, Inc.
548
device containing EO in an aqueous solution at 37 °C for
24 h. Following the analytical work, some biocompatibility
tests were conducted on EO sterilized polymers and the
aqueous extracts of EO sterilized devices.
MATERIALS AND METHODS
Materials
Two different polyurethanes, Pellethane 2363-75D (PU 75D)
and 2383-80A (PU 80A), and Nylon 66 were tested. Material
sheets 0.5 mm thick were cut into 10 ⫻45-mm strips with
five strips per specimen sample. Two types of electrophysi-
ology diagnostic electrode catheters were also tested. Both
types were from the same manufacturer (Bard), made of
cross-linked polyurethane (personal communication), and ap-
peared to be identical except for the connectors. However,
one catheter had embedded wires (designated solid or S) in
the shaft, whereas the other one was hollow with removable
wires (hollow or H). The wires in the hollow catheter samples
were removed before sterilization. The catheters were cut into
45-mm-long sections with nine sections composing a speci-
men sample.
EO Sterilization
Materials were sterilized on the weekend at the NIH Clinical
Center with the use of 10% EO at 54 °C, 65% RH for 130
min, followed by 12 h aeration at 132 °C. On Monday, after
2 days at room temperature, the samples were retrieved and
stored at ⫺70 °C until prepared for analysis. For resteriliza-
tion studies, samples were left at room temperature for 5 days
before resterilization with EO. Specimens were EO sterilized
0, 1, or 5 times.
Analytical Method
The EO stock standard used in this study was 50 mg/ml in
methanol (Suppelco 4-8838). The standard was then diluted
in water or methanol as needed. EO levels were determined
using the ISO method
7
. Headspace sampling is a method of
introducing volatile components, such as EO, from a liquid or
solid sample into a gas chromatograph. The vial is heated,
allowing the volatile compounds to go into the air (or head-
space) above the liquid or solid sample. After heating, a
gastight syringe is used to remove a portion of the air from
the headspace and inject the sample directly into the gas
chromatograph. A Hewlett-Packard headspace auto sampler
(HP 7694 oven 100 °C, loop 105 °C, vial equilibration time
15– 60 min, pressure: 0.5 min, loop fill: 0.15 min, loop
equilibration time 0.05 min), a HP gas chromatograph 5890
series II (inlet 105 °C, 30 m ⫻0.32 mm Omega-wax 320
capillary column; 30 °C 5 min, 20 °C/min to 100 °C, hold 15
min) and an FID detector (220 °C) were used.
Extraction Method
Samples were analyzed for total EO concentration. Solid
specimens were sealed in a vial and thermally extracted by
heating to 100 °C for 60 min, followed by GC residue
analysis of the headspace gas. Samples were repeatedly
heated and analyzed, with nitrogen purge or evacuation be-
tween cycles, until EO levels approached the limit of reliable
quantitation (approximately 5
g/g).
To evaluate bioavailability of EO residues, water, culture
media [RPMI-1640 with L-glutamine and 10% fetal bovine
serum (FBS)], and cottonseed oil were compared as extrac-
tion solutions. EO was added to each solution (500
g) and
kept at 37 °C for 24 h.
9
The following day, 1-ml aliquots were
removed and analyzed. Based on the results of the three
extraction solutions, water was deemed best, and all of the
materials were subsequently extracted in 13 ml of water for
24 h at 37 °C with 1 ml removed for GC residue analysis. The
hollow and solid catheter pieces used for liquid extraction had
a final surface area to extraction solution volume ratio of 1.96
cm
2
/ml. PU 75D, PU 80A, and Nylon 66 had a 3.5 cm
2
/ml
surface area to volume ratio.
8
Biocompatibility Testing
Apoptosis and Cytotoxicity. In one series of tests, 1 ml
of the water extracts for each material was placed into cell
culture with 2-ml Jurkat cells (a human lymphoma cell,
ATCC CRL 8163) to test viability and appearance of apo-
ptotic cells. In addition, EO standards (final concentration in
cell culture 0.2 to 83
g/ml) were prepared in water and
tested with these cells. Twenty-four hours after adding the
sample or EO standard, an aliquot of the cells was removed
and analyzed using a flow cytometer and LYSIS I-I software
(FACScan, Becton Dickenson, San Jose, CA), as previously
described.
10,11
Cells were analyzed according to the side-
scattering profile (proportional to cellular granularity) verses
the forward-scattering profile (proportional to the cellular
cross sectional area). Changes in the cell populations were
evaluated by gating on the normal cell population and com-
paring to the test groups over time. Apoptotic cells exhibit a
decrease in forward scatter (reduced cell size) and an increase
in side scatter (increased granularity) and can readily be
quantified with the use of a flow cytometer.
10 –13
Cytotoxicity Testing. The polymer PU 75D, which had
the highest EO residue, was tested for its effect on fibroblasts
in culture. EO levels for this particular lot of PU 75 D were
1540.7
g/g EO for exhaustively extracted and 458
g/g in
the water extracts. The protocol was conducted according to
ANSI/AAMI/ISO
14
using extracts and according to ASTM F
813
15
using direct contact tests. The PU 75D samples were
either used immediately after retrieval following the EO
sterilization and aeration protocol (detailed in EO sterilization
section) or were kept frozen at ⫺70 °C until use. The control
PU 75D needed to be sterilized by a means other than EO for
comparison; so control samples were sterilized by autoclav-
ing. Samples were cut to size before the sterilization cycles.
L929 fibroblast cells were grown to a confluent monolayer in
100 ⫻15-mm culture dishes with RPMI 1640 medium con-
taining 10% FBS. The medium was removed prior to addition
of the sample or extracts.
549RESIDUAL ETHYLENE OXIDE IN MEDICAL DEVICES
Direct Contact. The strips of PU 75D (from the same lot
as used in the cytotoxicity testing detailed above) were placed
directly on the cells. Fresh medium was then carefully added
to the dishes. The strips tended to float and those that floated
were carefully submerged with the pipette. The cultures were
examined at 24, 48, and 72 h for evidence of damage to the
cells in the monolayer.
Cytotoxicity of Medium Extracts. For examination of the
effect of extracts, PU 75D samples were placed into RPMI
1640 with FBS for 24 hours at 37 °C. The sample area to
volume ratio was 6 cm
2
/ml. At the end of 24 h, the extract
was withdrawn and placed on the monolayer of cells. The
monolayers were examined at 24, 48, and 72 h for evidence
of cytotoxicity.
Complement Activation. Whole complement activation
was screened in two steps, in accordance with ASTM Stan-
dard Practice F1984-99.
16
Briefly, 0.1 ml of a standard human
complement serum was placed on each of six polymer strips
that had not been exposed to EO, six strips previously ex-
posed to EO (stored at -70 °C until testing), were placed in six
glass tubes on ice, and in six glass tubes to be placed with the
polymer strips in 100% humidity at 37 °C. Following 1 h
incubation, all serum samples were transferred to cold glass
tubes on ice, diluted, and then tested for complement activity
by determining their capacity to lyse sheep red-blood cells
previously coated with a hemolytic antibody (indicated by
cell-free hemoglobin in test supernatant, tested for by absor-
bance at 405 nm).
RESULTS AND DISCUSSION
Resterilization
The amount of EO varied greatly for the different samples.
However, the number of sterilization cycles had little influ-
ence on the amount of EO residuals in the samples studied
(Figure 1).
Exhaustive Extraction
The values in Figure 2 are the cumulative EO values over the
accumulated extraction time. Repeated thermal extraction of
some specimens was necessary to obtain the total amount of
EO. PU 75D had much higher EO residue, while PU 80A had
barely detectable levels (less than 10
g/g, data not shown).
After EO sterilization and aeration, repeated thermal desorp-
tion of some materials was necessary to obtain total EO
residues; the Nylon 66 and PU 75D were thermally extracted
8 times at 60 min at 100 °C to extract most of the EO (Figure
2). PU 75D is harder and more crystalline than PU 80A.
Previous studies of EO residues in polymers demonstrate that
the more crystalline polymer have higher residue,
17–19
poly-
mers with increased chain length desorb EO slower,
19
hard-
ened polymers release EO slowly,
5
while glassy polymers
retain the lowest amounts.
17
Also, highly porous materials
tend to have higher EO values
20
due to low diffusion coef-
ficients and high solubility. Tensile strength testing following
EO sterilization for PU 75D, PU 80A, and Nylon 66 has been
reported.
21
After EO sterilization, PU 75D showed a large
loss of tensile strength, Nylon 66 a small but significant
increase, while PU 80A did not change. These data may
indicate that for those materials that retain larger quantities of
EO, larger changes in material properties, such as strength,
might occur.
The solid and hollow catheter pieces also had significantly
different amounts of residual EO. This was a bit surprising, as
both devices were purchased from the same manufacturer
with the only apparent differences being the connectors and
whether the wires were embedded or removable. The solid
catheter needed to be thermally desorbed repeatedly, just like
Figure 1. Resterilization effect. Materials were resterilized with EO
one (1 ⫻) or five (5 ⫻) times. There was no significant difference in
residue levels between sterilizing once or five times for the polymers
and devices used in this study. PU is polyurethane; N, nylon, S, solid
catheter; H hollow catheters.
Figure 2. Cumulative EO thermally extracted from PU 75D, Nylon 66,
and solid catheter pieces. Samples were thermally extracted eight
times at 60 min at 100 °C then analyzed until EO levels were at the
limit of detection. Both PU 80A and the hollow catheter had very low
levels of EO (less than 10
g/g and 10.4
g/g, respectively; data not
shown).
550 LUCAS ET AL.
the Nylon 66 and the PU 75 D, whereas the hollow catheter
pieces had low EO residues (10.2
g/g).
Extraction Solutions
Figure 3 shows a comparison of three candidate solutions for
extracting EO sterilized medical devices and polymers to
determine residues that are bioavailable. The culture medium
with FBS had much less extractable EO, as the EO probably
reacted with the proteins in the solution (Figure 3). The
control culture medium had some substances that coeluted
with EO. Water and oil did not have any coeluting peaks;
however, the oil presented some significant reproducibility
problems. Because EO is a relatively polar molecule, it did
not dissolve in the oil and was not dispersed uniformly,
resulting in large standard deviations (see Figure 3). Water
showed good reproducibility and a reasonable signal; there-
fore, it was chosen as the extraction solution.
Total EO versus Bioavailable EO. Comparison of EO
residue levels from exhaustive extraction with the levels
obtained from water extraction (Figure 4) illustrates that for
some polymers (Nylon, PU80A, hollow catheters) there is no
significant difference, whereas for others (PU75D, solid cath-
eters) there is up to an eightfold difference. This is likely
related to the microstructure of the polymeric material. Pre-
vious studies have shown that materials with increasing crys-
tallinity will retain more EO, whereas softer flexible materials
will retain less.
17,18
Presumably, the EO retention difference
between the catheters is due to the physical structure. The
hollow catheters had a much larger surface area for EO
diffusion.
Biocompatibility
Apoptosis and Cytotoxicity. No significant difference
was seen between control cells and those with the water
extracts from PU 75D, Nylon 66, PU 80A, or solid or hollow
catheter pieces (data not shown). The current limit for EO
exposure from most medical devices is 250 ppm,
6,7
and even
adding 250
g of the EO standard directly to the cells showed
no changes in viability or number of apoptotic cells. This is
probably due to two factors. First, a cold solution cannot be
added directly to the cells. In warming the water up to room
temperature, EO becomes a gas (boiling point 10.4 °C).
Second, in cell-culture medium, there are large amounts of
proteins, lipids, and other biological molecules in solution.
EO is a reactive alkylating agent; it kills microbes by adding
alkyl groups to DNA, RNA, and proteins. This also makes
EO a toxin for human beings. Before a significant amount of
EO can reach the cells to affect them, most of the EO had
probably reacted first with components in the biological
media.
Direct Contact and Indirect Extraction Assays. For the
PU 75D strips used in these assays, there was an average of
1540.7
g/g EO in the exhaustively extracted sample and 458
g/g EO in water extracts (data not shown). There was no
evidence of damage to the cell monolayer by direct contact
with the EO-sterilized PU 75D strips or by direct contact with
the autoclaved strips. Cells grew up to and onto the strips.
Similarly, the extracts did not cause any damage to the cells.
The extracts themselves were clear, pH did not change, and
they supported cell growth. Previous reports of different
materials have shown a small effect on L929 cells at this
level
22
(agar overlay method).
Complement Assay. Although the PU 75D EO sterilized
strip did activate complement, it was not significantly more
than PU 75D not treated with EO. Although these specific
biocompatibility tests did not generate a positive response,
the toxicity, carcinogenicity, mutagenicity, and teratogenicity
of EO have been well documented.
3
Figure 4. Total EO compared to bioavailable EO. Comparison of the
total amount of thermally extracted EO to that which is bioavailable
(extracted in water). Only PU 75D (a polyurethane) and solid catheter
pieces (solid) showed a significant difference between total EO and
bioavailable EO. PU 75D had approximately 8 times more EO in the
total extract, whereas the solid catheter had 5.5 times more. Nylon 66,
hollow catheter pieces (hollow), and polyurethane 80A (PU80A)
showed no significant differences.
Figure 3. Extraction solution effect. Reproducibility and recovery of
EO (relative to water) in different extraction solutions. 500
g/ml of EO
in cottonseed oil, water, and media were incubated at 37 °C for 24 h,
then analyzed. Media had some coeluting interferences and the oil
had reproducibility problems. Water was used for all subsequent
extractions.
551RESIDUAL ETHYLENE OXIDE IN MEDICAL DEVICES
CONCLUSIONS
Resterilization with EO has been reported to increase the total
EO residues in some materials
5
. However, for the devices and
polymers used in this study, no significant differences were
seen, even after five resterilization cycles. EO residues in
medical devices and polymers depend on the type and size of
the material. Some materials retain little EO residues, such as
PU 80A and hollow catheters, whereas others retain much
larger amounts, such as PU 75 D and Nylon 66. The choice
of extraction solutions affects the amount of EO detected.
The bioavailability of EO from medical devices and polymers
when compared to the total amount of extractable EO varied
widely. For some materials (PU 75D) EO is much less
extractable in water than was found from exhaustive extrac-
tion (eightfold less). Other polymers (PU 80A, Nylon 66) had
little difference between the amount of total EO and that
which is bioavailable. Adverse bioeffects of EO were not
seen in this study. There was no significant difference be-
tween EO treated PU 75D and untreated PU 75D in comple-
ment activation. Direct and indirect cell culture biocompat-
ibility and viability assays showed no effects. This is possibly
due to a number of factors: (a) EO can diffuse out slowly, (b)
EO reacts with medium components, and (c) EO is a gas at
room temperature (10.4 °C boiling point) and may not even
be in solution for biological testing. However, the toxicity of
EO in vitro and in vivo is well documented.
2– 4
The work presented here illustrates that EO levels and
effects are highly dependent on the type of material used.
Residual EO, the amount of EO that is bioavailable, and the
effect of EO in vitro must be considered for each type of
material or device.
The authors would like to thank Walter Reed Hospital for pro-
viding the catheters and NIH for EO sterilization.
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