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. Comparison 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 biocompatibility tests were run on extracts and devices, but no significant effects were observed.
Discover the world's research
- 20+ million members
- 135+ million publications
- 700k+ research projects
Join for free
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.
549 RESIDUAL 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.
551 RESIDUAL 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.
REFERENCES
1. MacNeil JR, Glaser ZR. Comparison of health care-based
sterilization technologies: Safety, efficacy, and economics.
J Healthcare Safety, Compliance & Infection Control 1997;
91–106.
2. International Agency for Research on Cancer. Ethylene Oxide.
IARC monograph on evaluation of carcinogenic risk of chem-
icals to humans; 1985. vol. 36, pp 189 –226.
3. Thier R, Bolt HM. Carcinogenicity and genotoxicity of ethylene
oxide: New aspects and recent advances. Crit Rev Toxicol
2000;30:595– 608.
4. World Health Organization. Ethylene oxide. Environmental
Health Criteria 55, World Health Organization. Geneva: WHO;
1985.
5. Buben I, Melierikoia V, Novona N, Sviakoa R. Problems asso-
ciated with sterilization using ethylene oxide. Residues in
treated materials. Cent Eur J Public Health 1999;7:197–202.
6. Association for the Advancement of Medical Instrumentation.
Technical Information Report 19. Guidance for ANSI/AAMI/
ISO 10993-7: 1995. Biological evaluation of medical devices—
Part 7: Ethylene oxide sterilization residuals. Baltimore, MD:
Association for the Advancement of Medical Instrumentation;
AAMI TIR 19:1998.
7. International Standards Organization. Biological evaluation of
medical devices—Part 7: Ethylene oxide sterilization residuals.
ANSI/AAMI/ISO 10993-7. Baltimore, MD: Association for the
Advancement of Medical Instrumentation; 1995.
8. ASTM Standard Practice F619-02: Standard practice for extrac-
tion of medical plastics. West Conshohocken, PA: American
Society for Testing and Materials; 1997. pp F619 –F679.
9. International Standards Organization. Biological evaluation of
medical devices - Part 12: Sample preparation and reference
materials. ANSI/AAMI/ISO 10993-12. Baltimore, MD: Asso-
ciation for the Advancement of Medical Instrumentation; 1996.
10. Godar DE, Miller SA, Thomas, DP. Immediate and delayed
apoptotic cell death mechanisms: UVA verses UVB and UVC
radiation. Cell Death Differen 1994;1:59 – 69.
11. Godar DE, Lucas AD. Spectral dependence of UV-induced
immediate and delayed apoptosis: The role of membrane and
DNA damage. Photochem Photobiol 1995;62:108 –113.
12. Dive C, Gregory CD, Phipps DJ, Evans DL, Milner AE, Wyllie
AH. Analysis and discrimination of necrosis and apoptosis
(programmed cell death) by multiparameter flow cytometry.
Biochem Biophys Acta 1992;1133:275–285.
13. Darzynkiewicz Z, Bruno S, Del Bino G, Gorczyca W, Hotx
MA, Lassota P, Tranganos F. Features of apoptotic cells mea-
sured by flow cytometry. Cytometry 1992;13:795– 808.
14. International Standards Organization. Biological evaluation of
medical devices—Part 5: Tests for cytotoxicity, in vitro meth-
ods. ANSI/AAMI/ISO 10993-5. Baltimore, MD: Association
for the Advancement of Medical Instrumentation; 1999.
15. ASTM Standard Practice F 813-01: Standard practice for direct
contact cell culture evaluation of materials for medical devices.
Annual Book of ASTM Standards. West Conshohocken, PA:
American Society for Testing and Materials; 2001. vol. 13.01.
16. ASTM Standard Practice F 1984–99: Standard practice for
testing for whole complement activation in serum by solid
materials. Annual Book of ASTM Standards 2001, volume
13.01
17. Gilding, DK, Reed AM, Baskett SA. Ethylene oxide steriliza-
tion: effect of polymer structure and sterilization conditions on
residue levels. Biomat 1980;1:145-148.
18. Vink P, Pleijsier K. Aeration of ethylene oxide-sterilized poly-
mers. Biomat 1986;7:225-230.
19. Guess WL. Residual ethylene oxide and reaction products in
polymers. Bull Parenteral Drug Assoc 1970;24:68 –75.
20. Nair P. Currently practiced sterilization methods—Some inad-
vertent consequences. J Biomater Appl 1995;10:121–135.
21. Brown SA, Merritt K, Woods TO, McNamee SG, Hitchins VM.
Effects of different disinfection and sterilization methods on the
tensile strength of materials used for single-use devices. Biomed
Instrum Tech. 2002;36:23–27.
22. McGunnigle RG, Renner JA, Romano SJ, RA Abodeely. Re-
sidual ethylene oxide: Levels in medical grade tubing and
effects on an in vitro biological system. J Biomed Mater Res
1975;9:273–283.
552 LUCAS ET AL.
... Os dispositivos médicos considerados críticos (aqueles que são introduzidos em áreas estéreis do corpo) e termossensíveis (os que não resistem aos métodos de esterilização a altas temperaturas), para serem reutilizados, necessitam de métodos de esterilização a baixa temperatura, a exemplo da esterilização a óxido de etileno (OE), plasma de peróxido de hidrogênio, vapor de formaldeído e ozônio [8][9] . Entre esses métodos, a esterilização a OE é o mais antigo e considerado padrão-ouro, pela alta difusibilidade e potência do gás esterilizante, contudo é também o mais tóxico dos métodos esterilizantes [8][9][10][11][12][13][14][15][16][17][18][19] . ...
... Os produtos esterilizados pelo OE podem apresentar resíduos tóxicos (etilenocloridrina e etilenoglicol) que, se não removidos, são passíveis de acarretar danos aos pacientes usuários desses produtos, aos profissionais manipuladores e ao meio ambiente. Desse modo, é imperativo que tais PPS sejam submetidos a um processo denominado de "aeração" para remover os resíduos tóxicos [8][9][10][11][12][13][14][15][16][17][18][19][20] . ...
... Nesse sentido, a aeração mecânica, não mencionada por essa norma, constitui o padrão-ouro da aeração de produtos esterilizados por OE, contudo esse processo também requer controles de temperatura e fluxos de ar dentro da câmara Há consenso entre autores de que a duração da aeração depende de fatores já descritos nesta revisão e, entre esses produtos confeccionados à base de cloreto de polivinil (PVC), poliestireno e borrachas são os que mais absorvem OE. Assim, não existe um tempo padrão recomendado para aeração de todos os dispositivos esterilizados por esse agente [8][9][10][11][16][17]20,30,40,23,26,18,35 . ...
- Eliana Auxiliadora Magalhães Costa
Objetivos: Descrever níveis residuais aceitáveis de óxido de etileno em dispositivos médicos, analisar processos de aeração recomendados e compará-los com a regulaçãobrasileira. Método: Revisão integrativa da literatura, com descritores específicos, sem restrição de ano de publicação. Busca dos dados entre outubro e novembro de 2019,que resultou em 34 estudos incluídos no estudo. Resultados: A regulação brasileira vigente está desatualizada em relação à classificação de produtos, à determinação de valoresde resíduos tóxicos de óxido de etileno em dispositivos médicos e aos processos recomendados para a aeração desses produtos, podendo contribuir para riscos de eventos adversospara pacientes usuários de dispositivos inadequadamente aerados, e, consequentemente, urge sua atualização. Conclusão: As lacunas desse marco regulatório beneficiamindiretamente as empresas que terceirizam a esterilização a óxido de etileno ao omitir controles essenciais para a segurança do paciente exposto a possíveis resíduos tóxicosde óxido de etileno, favorecer práticas inseguras de esterilização de produtos para saúde, além de dificultar o controle de serviço de saúde pelas vigilâncias sanitárias do país.
... While these are widely used techniques, the exploration of possible scaffold damage from these techniques should be investigated. For example, ethanol is drying to the scaffolds and could fracture the intricate tissue structure, while ethylene oxide (EO) has been known to deposit a toxic residue [71]. ...
The decellularization of plant-based biomaterials to generate tissue-engineered substitutes or in vitro cellular models has significantly increased in recent years. These vegetal tissues can be sourced from plant leaves and stems or fruits and vegetables, making them a low-cost, accessible, and sustainable resource from which to generate three-dimensional scaffolds. Each construct is distinct, representing a wide range of architectural and mechanical properties as well as innate vasculature networks. Based on the rapid rise in interest, this review aims to detail the current state of the art and presents the future challenges and perspectives of these unique biomaterials. First, we consider the different existing decellularization techniques, including chemical, detergent-free, enzymatic, and supercritical fluid approaches that are used to generate such scaffolds and examine how these protocols can be selected based on plant cellularity. We next examine strategies for cell seeding onto the plant-derived constructs and the importance of the different functionalization methods used to assist in cell adhesion and promote cell viability. Finally, we discuss how their structural features, such as inherent vasculature, porosity, morphology, and mechanical properties (i.e., stiffness, elasticity, etc.) position plant-based scaffolds as a unique biomaterial and drive their use for specific downstream applications. The main challenges in the field are presented throughout the discussion, and future directions are proposed to help improve the development and use of vegetal constructs in biomedical research.
... For example, γ radiation generates free radicals that can cause tissue damage (Ries et al., 1996;Gorna and Gogolewski, 2003;Mendes et al., 2007). Similarly, ethylene oxide and its derivatives have been reported to accumulate in some materials, potentially increasing teratogenicity risk (Lucas et al., 2003;Mendes et al., 2007). As previously mentioned, the investigator should characterize and validate each sterilization technique for each FSCV sensor type, composition, and geometry to ensure patient safety, in addition to signal accuracy. ...
Fast Scan Cyclic Voltammetry (FSCV) has been used for decades as a neurochemical tool for in vivo detection of phasic changes in electroactive neurotransmitters in animal models. Recently, multiple research groups have initiated human neurochemical studies using FSCV or demonstrated interest in bringing FSCV into clinical use. However, there remain technical challenges that limit clinical implementation of FSCV by creating barriers to appropriate scientific rigor and patient safety. In order to progress with clinical FSCV, these limitations must be first addressed through (1) appropriate pre-clinical studies to ensure accurate measurement of neurotransmitters and (2) the application of a risk management framework to assess patient safety. The intent of this work is to bring awareness of the current issues associated with FSCV to the scientific, engineering, and clinical communities and encourage them to seek solutions or alternatives that ensure data accuracy, rigor and reproducibility, and patient safety.
... Gas sterilization with ethylene oxide was considered, however, we were concerned about toxic residues remaining in the treated material. 35 Heatbased methods for sterilization were deemed inappropriate as our materials were not heat or stream resistant. ...
Scaffold guided breast tissue engineering has the potential to transform reconstructive breast surgery. Currently, there is a deficiency in clinically relevant animal models suitable for studying novel breast tissue engineering concepts. To date, only a small number of large animal studies have been conducted and characterisation of these large animal models is poorly described in the literature. Addressing this gap in the literature, this publication comprehensively describes our original porcine model based on the current published literature and the experience gained from previous animal studies conducted by our research group. In a long-term experiment using our model, we investigated our scaffold guided breast tissue engineering approach by implanting 60 additively manufactured bioresorbable scaffolds under the panniculus carnosus muscle along the flanks of 12 pigs over 12 months. Our model has the flexibility to compare multiple treatment modalities where we successfully investigated scaffolds filled with various treatments of immediate and delayed fat graft and augmentation with platelet rich plasma (PRP). No wound complications were observed using our animal model. We were able to grow clinically relevant volumes of soft tissue, which validates our model. Our preclinical large animal model is ideally suited to assess different scaffold or hydrogel driven soft tissue regeneration strategies.
... Ethylene oxide (EO) is the sterilization technique generally used for polymer-based medical devices, as it can be performed at low-temperatures, which leads to minimal changes in molecular weight compared to other conventional methods [19][20][21]. The major concern with EO sterilization is the presence of toxic residues that can reside on the implant after the process [22][23][24]. In order to remove EO residues such as ethylene glycol and ethylene hydrochloride, an aeration of EO sterilized medical devices must be performed which leads to a lengthening of the final process [25,26]. ...
Although bioabsorbable polymers have garnered increasing attention because of their potential in tissue engineering applications, to our knowledge there are only a few bioabsorbable 3D printed medical devices on the market thus far. In this study, we assessed the processability of medical grade Poly(lactic-co-glycolic) Acid (PLGA)85:15 via two additive manufacturing technologies: Fused Filament Fabrication (FFF) and Direct Pellet Printing (DPP) to highlight the least destructive technology towards PLGA. To quantify PLGA degradation, its molecular weight (gel permeation chromatography (GPC)) as well as its thermal properties (differential scanning calorimetry (DSC)) were evaluated at each processing step, including sterilization with conventional methods (ethylene oxide, gamma, and beta irradiation). Results show that 3D printing of PLGA on a DPP printer significantly decreased the number-average molecular weight (Mn) to the greatest extent (26% Mn loss, p < 0.0001) as it applies a longer residence time and higher shear stress compared to classic FFF (19% Mn loss, p < 0.0001). Among all sterilization methods tested, ethylene oxide seems to be the most appropriate, as it leads to no significant changes in PLGA properties. After sterilization, all samples were considered to be non-toxic, as cell viability was above 70% compared to the control, indicating that this manufacturing route could be used for the development of bioabsorbable medical devices. Based on our observations, we recommend using FFF printing and ethylene oxide sterilization to produce PLGA medical devices.
... Ethylene Oxide (EtO) is known to be a human carcinogen, however, it is commonly used to sterilise medical devices [38]. Contact with the skin, for example, can cause irreversible toxic effects [39]. ...
The current COVID-19 pandemic has resulted in an immense and unforeseen increase in demand for personal protective equipment (PPE) for healthcare workers worldwide. Amongst other products, respirator masks are crucial to protect the users against transmission of the virus. Decontamination and reuse of the existing stock could be a solution to the shortage of new respirators. Based upon existing studies, it was found that (I) a solid quality control method is essential to test product reuse, (II) in-depth evaluation of the different parts of the filtering facepiece respirator (FFR) should be considered, and (III) communication of the reuse cycle is essential to take track of the amount of reuse, as this is limited to ensure quality. The goal of this paper is two-fold. First, we identify the impact of decontamination on the different parts of the FFRs and how the quality control should be performed. Two different types of FFRs are analysed within this paper, resulting in the recommendation of combining quantitative respirator mask fit testing with a thorough sensory evaluation of decontaminated FFRs to qualify them for reuse. Secondly, the possibilities of communication of this reuse to the eventual user are mapped through in-depth reasoning.
... Concern of EtO gas residuals in the scaffolds has always precluded this prospect. Current methods for testing EtO residuals involve extraction in water, so the cytotoxicity elution assay would be an appropriate method to determine the effect of residuals on cell viability 174 . Most importantly, EtO sterilization would substantially simplify the BVM fabrication process; scaffolds could be secured in bioreactors before sterilization, so only media would need to be added to the bioreactors and the flow circuit assembled before starting conditioning. ...
- Conor Charles Hedigan
Blood vessel mimics (BVMs) are simple tissue engineered blood vessel constructs intended for preclinical testing of vascular devices. This thesis developed and implemented methods to characterize two of these components. The first aim of this thesis investigated the effect of cell culture duration and flow conditions on endothelial cell gene expression, especially regarding endothelial-to-mesenchymal transition (EndMT). A trend of decreased endothelial marker gene expression and increased mesenchymal marker gene expression would indicate EndMT. qPCR analysis revealed that increased cell culture duration did not result in EndMT, and in fact increased endothelial marker expression as cell culture duration increased. Disturbed flow conditions decreased endothelial marker and increased mesenchymal marker expression relative to static culture. The second aim of this thesis developed methods to determine cytotoxicity of, and endothelial cell adhesion to, novel BTEAC salt scaffolds. Immunostaining was used to visualize these scaffold effects. The cytotoxicity elution assay showed that BTEAC salt scaffolds were not more cytotoxic than the standard PLGA scaffold. Direct contact assays spanning several timepoints also found that BTEAC salt scaffolds were not more cytotoxic than standard scaffolds but had higher endothelial cell adhesion and coverage than standard scaffolds. Overall, this thesis developed and implemented methods to characterize the endothelial cells used in the BVM model.
In pathologies of the esophagus such as esophageal atresia, cancers and caustic injuries, methods for full thickness esophageal replacement require the sacrifice of healthy intra-abdominal organs such as the stomach and the colon. These methods are associated with high morbidity, mortality and poor functional results. The reconstruction of an esophageal segment by tissue engineering (TE) could answer this problem. For esophageal TE, this approach has been explored mainly by a combination of matrices and cells. In this chapter, we will discuss the studies on full organ esophageal decellularization, including the animal models, the methods of decellularization and recellularization.
- Mi Suk Noh
- Suk Hee Jung
- Ohryun Kwon
-
![Bong-Hyun Jun]()
Hydrogen-peroxide-based low-temperature sterilization is a new sterilization technology for temperature-dependent medical devices. The effect of the process parameters of hydrogen-peroxide-based sterilizer on the sterilization performance of process challenge devices (PCDs) needs to be investigated. Sterilant amount, operating temperature, vacuum pressure, diffusion time, and chamber loading of the sterilizer on the sterilization performance of PCDs were adjusted. Seven PCDs with various morphologies and material containing biological indicators (BI) (EZTest, Geobacillus stearothermophilus) were used to evaluate the sterilization performance. The sterilization success rates of PCDs were 86, 71, and 57% with controlled temperature and pressure, diffusion time, and sterilant volume injection, respectively. The PCD material and structure also obviously affected sterilization performance. The sterilization of PCD A is the least successful for all parameters. Meanwhile, the sterilization of PCD B was influenced by the diffusion time and the sterilant injection volume. PCD B and PCD C were successfully sterilized by controlling the temperature and pressure. The weights and volume of the sterilization loading chamber resulted in a different sterilization performance. Sterilization performances of PCD 1, PCD 2, and PCD 3 were <70, <90, and 100%, respectively. Sterilant volume, sterilant diffusion time, pressure, temperature, PCD types, and chamber loading were proven to be important process parameters of sterilizer that affect the sterilization performance of vaporized-hydrogen-peroxide-based sterilizers.
- R G Mc Gunnigle
- J A Renner
- S J Romano
- R. A. Abodeely
The level of residual ethylene oxide after sterilization was evaluated as a function of aeration time for three medical grade tubings. Toxicity resulting from residual ethylene oxide was determined in an in vitro tissue culture system utilizing L-cells. The absorption and desorption of ethylene oxide from poly(vinyl chloride) and polyether-polyurethane tubing were similar. In contrast, silicone tubing absorbed 85% less ethylene oxide. The time required for desorption of residual ethylene oxide was 2 hr for silicone tubing and 7 to 8 hr for poly(vinyl chloride) and polyether-polyurethane tubing. Tubing samples containing 1,500 ppm or more residual ethylene oxide elicited toxic tissue culture reactions whereas samples containing 900 ppm or less showed no toxic tissue culture response.
-
![Zbigniew Darzynkiewicz]()
- Bruno SG
- G Del Bino
- F Traganos
The present review describes several methods to characterize and differentiate between two different mechanisms of cell death, apoptosis and necrosis. Most of these methods were applied to studies of apoptosis triggered in the human leukemic HL-60 cell line by DNA topoisomerase I or II inhibitors, and in rat thymocytes by either topoisomerase inhibitors or prednisolone. In most cases, apoptosis was selective to cells in a particular phase of the cell cycle: only S-phase HL-60 cells and G0 thymocytes were mainly affected. Necrosis was induced by excessively high concentrations of these drugs. The following cell features were found useful to characterize the mode of cell death: a) Activation of an endonuclease in apoptocic cells resulted in extraction of the low molecular weight DNA following cell permeabilization, which, in turn, led to their decreased stainability with DNA-specific fluorochromes. Measurements of DNA content made it possible to identify apoptotic cells and to recognize the cell cycle phase specificity of the apoptotic process. b) Plasma membrane integrity, which is lost in necrotic but not apoptotic cells, was probed by the exclusion of propidium iodide (PI). The combination of PI followed by Hoechst 33342 proved to be an excellent probe to distinguish live, necrotic, early- and late-apoptotic cells. c) Mitochondrial transmembrane potential, assayed by retention of rhodamine 123 was preserved in apoptotic but not necrotic cells. d) The ATP-dependent lysosomal proton pump, tested by the supravital uptake of acridine orange (AO) was also preserved in apoptotic but not necrotic cells. e) Bivariate analysis of cells stained for DNA and protein revealed markedly diminished protein content in apoptotic cells, most likely due to activation of endogenous proteases. Necrotic cells, having leaky membranes, had minimal protein content. f) Staining of RNA allowed for the discrimination of G0 from G1 cells and thus made it possible to reveal that apoptosis was selective to G0 thymocytes. g) The decrease in forward light scatter, paralleled either by no change (HL-60 cells) or an increase (thymocytes) of right angle scatter, were early changes during apoptosis. h) The sensitivity of DNA in situ to denaturation, was increased in apoptotic and necrotic cells. This feature, probed by staining with AO at low pH, provided a sensitive and early assay to discriminate between live, apoptotic and necrotic cells, and to evaluate the cell cycle phase specificity of these processes. i) The in situ nick translation assay employing labeled triphosphonucleotides can be used to reveal DNA strand breaks, to detect the very early stages of apoptosis.(ABSTRACT TRUNCATED AT 400 WORDS)
- Caroline Dive
-
![Chris Gregory]()
- Donna J. Phipps
- AH Wyllie
Necrosis and apoptosis are two distinct modes of cell death which differ in morphology, mechanism and incidence. Membrane disruptants, respiratory poisons and hypoxia cause ATP depletion, metabolic collapse, cell swelling and rupture leading to inflammation. These are typical features of necrosis. Apoptosis plays a crucial role in embryogenesis and development and is also prevalent in tumours. It is characterised by cell shrinkage, chromatin condensation and systematic DNA cleavage. Apoptotic cells are rapidly engulfed by phagocytes, thus preventing inflammatory reaction to degradative cell contents. In vivo, apoptosis is almost impossible to quantify due to problems of heterogeneity and the short half-life of an apoptotic cell. In vitro, mechanistic studies are further complicated by a late phase of apoptosis where the cell membrane becomes permeable to vital dyes and which occurs in the absence of phagocytes. Here we describe a novel and rapid multiparameter flow cytometric assay which discriminates and quantifies viable, apoptotic and necrotic cells via measurement of forward and side light scatter (proportional to cell diameter and internal granularity, respectively) and the DNA-binding fluorophores Hoechst 33342 and propidium. It is anticipated that mechanistic studies of apoptosis in a variety of cell types will greatly benefit from this mode of analysis.
- P Vink
- K Pleijsier
The ethylene oxide (EO) content of 17 polymers sterilized with 100% EO under conditions normally used in practice was determined as a function of aeration time, and for some polymers also as a function of sample thickness. The determination of the amount of residual EO has been carried out by gas chromatography, applying Discontinuous Gas Extraction and Head Space analysis. Generally, aeration was confirmed to be a diffusion-controlled process. Diffusion coefficients of EO for the investigated materials were determined from the rates of desorption. For a number of materials the EO content appeared to be well above the levels for EO which currently are considered to be safe, even after aeration for 15 d. For a reliable determination of aeration times required for EO-sterilized medical devices, the type and in particular also the thickness of the material from which the device is made should be considered.
The phototoxicity of each waveband region of UV radiation (UVR), i.e., UVA (320-400 nm), UVB (290-320 nm) and UVC (200-290 nm), was correlated with an apoptotic mechanism using equilethal doses (10% survival) on murine lymphoma L5178Y-R cells. Apoptosis was qualitatively monitored for DNA "ladder" formation (multiples of 200 base pair units) using agarose gel electrophoresis, while the percentages of apoptotic and membrane-permeabilized cells were quantified over a postexposure time course using flow cytometry. The UVA1 radiation (340-400 nm) induced both an immediate (< 4 h) and a delayed (> 20 h) apoptotic mechanism, while UVB or UVC radiation induced only the delayed mechanism. The role of membrane damage was examined using a lipophilic free-radical scavenger, vitamin E. Immediate apoptosis and membrane permeability increased in a UVA1 dose-dependent manner, both of which were reduced by vitamin E. However, vitamin E had little effect on UVR-induced delayed apoptosis. In contrast, the DNA damaging agents 2,4- and 2,6-diaminotoluene exclusively induced delayed apoptosis. Thus, immediate apoptosis can be initiated by UVA1-induced membrane damage, while delayed apoptosis can be initiated by DNA damage. Moreover, the results suggest that immediate and delayed apoptosis are two independent mechanisms that exist beyond the realm of photobiology.
-
![Prabha Damodaran Nair]()
Currently used sterilization techniques such as ethylene oxide, gamma irradiation, and steam sterilization could introduce inadvertent consequences, especially in polymeric materials. These could have far-reaching effects on the biocompatibility of the materials. Some of these consequences are reviewed and a typical example of the effect of steam sterilization on the properties and biocompatibility of polyethylene terephthalate is discussed.
- I. Buben
- V. Melicherčíková
- N. Novotná
- R. Svitáková
The paper deals with problems associated with reduction of undesirable effects of ethylene oxide in polymers in medical devices on the patient's health. The authors explain the need of careful elaboration and validation of the sterilization and aeration process incl assessment of ethylene oxide (EO) residues. The authors investigated the effect of the type of material and conditions of sterilization and aeration on the assessed EO concentration. For research of the behaviour of different polymers in the sterilization process model sterilizations of actual items of medical devices with a known composition proved more suitable than assessment in medical devices from medical institutions. The main conclusions of the investigation were a classification of polymers into those suitable and unsuitable for sterilization or resterilization, and attention was also drawn to poor reproducibility of results in old sterilizers, in particular those lacking effective aeration in aerators.
Source: https://www.researchgate.net/publication/10660163_Residual_Ethylene_Oxide_in_Medical_Devices_and_Device_Material
Posted by: malcolmkilee0193641.blogspot.com
