Ethylene oxide (EO) gas is commonly used to sterilize medical devices. A major concern is the amount of residue that may remain on or in the device and be available in the body. Some standards (ASTMF619 and ISO 10993-12) recommend using two different extraction solutions (one polar, one nonpolar), for sample preparation prior to testing medical devices. However, ISO 10993-7 recommends water to process medical devices to determine EO residual levels. To address this, EO residual levels were examined in different extraction solutions. EO residual levels from devices and materials extracted with different solutions were evaluated. Results from this study indicate little difference between extraction solutions of water, cell culture media, and serum (less than 30% difference). Given the increased cost and increased background noise of media or serum over water, using only water to process medical devices and materials for EO residues appears adequate.
Ethylene oxide (EO) gas is routinely used to sterilize medical devices that can't tolerate high temperature or radiation and also because of its good material compatibility, simplicity, and relatively low cost.1–3 However, EO and some of its degradation products are carcinogenic and mutagenic.4–7 Thus, there are allowable limits for medical devices that will be used in patients.8 Current allowable limits for EO exposure from medical devices are 250 ppm (recommended level from the Association for the Advancement of Medical Instrumentation (AAMI) Technical Information Report 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.8,9 The major concern in the analysis of EO residuals is accurately determining the amount of EO to which a person might be exposed. In order to determine the EO residuals on a device, one must extract and measure the residuals. Water extraction at 37°C is recommended by AAMI8; however, there is little information on other extraction solutions for EO residual determination.
Water, culture media (Roswell Park Memorial Institute Medium [RPMI]-1640 with L-glutamine and 10% fetal bovine serum [FBS]), and cottonseed oil were previously compared as extraction solutions.10 The culture media signal response for EO was about 65% that of water. The response for cottonseed oil was the same as the water, but had poorer reproducibility.10 Because standards recommend using at least two different extraction solution of different polarity,11,12 the evaluation of different physiological solutions to examine residual EO levels was conducted. Using the method developed by the American National Standards Institute/AAMI/International Standards Organization (ISO)8 for headspace analysis of EO residues, EO was added to a variety of solutions to evaluate signal response. Additionally, materials and devices sterilized with EO were extracted in water, cell culture media, and FBS, and the residual concentrations were compared.
Materials and Methods
EO stock standard used in this study was 50 mg/mL in methanol (Suppelco 4-8838). Cell culture media used were RPMI-1640, both with and without phenol red, and CO2 independent media (Gibco Laboratories, Grand Island, NY). Heat-inactivated FBS (Sigma Chemical Co., St. Louis, MO) and Ringers lactate (Moore Medical Corp, New Britain, CT) were purchased. To evaluate signal response of EO residues, water, RPMI-1640 with no dye, RPMI with dye and 10% FBS, CO2 independent media with 10% FBS, and Ringers lactate with 5% FBS were compared. EO was added to each solution (0-50 μg) and analyzed to assess the signal response relative to water. As this was a screening experiment, samples were run in singlet and analyzed immediately.
Items that were sterilized with EO to evaluate different extraction solutions were 35 mm photographic film, two different angiographic catheters, film negatives, one peripheral balloon dilation catheter, and an electrophysiology catheter. Prior to EO sterilization, items were cut into small sizes (less than 5 cm), weighed, and surface area was calculated. Following EO sterilization, triplicate samples of the devices and materials were processed in water, CO2 independent media, and FBS at 37°C for 24 hours. Because the EO standard is available in methanol, the same amount of methanol was added to the device extracts to account for any cosolvent effect.
The samples were sterilized at the National Institutes of Health using Oxyfume 2002 Sterilant Mixture (EO 10%; chlorodifluoromethane (HCFC-22) 27%; chlorotetrafluoroethane (HCFC-124) 63%). The items require a 14- to 16-hour cycle. Sterilizer parameters are maximum temperature: 131.0°F, minimum temperature: 127.0°F; sterilization time: 2 hours and 10 minutes; pressure (psig): 2.6 in Hg; aeration time: 12 hours; pressure (psig): 25.5 in Hg. The samples were stored at −70°C until prepared for analysis.
EO levels were determined using the ISO method.8 A Hewlett-Packard (HP) headspace auto sampler (HP 7694; oven: 100°C; loop: 105°C: vial equilibration time: 15–60 minutes; pressure: 0.5 minutes; loop fill: 0.15 minutes; loop equilibration time: 0.05 minutes), an HP Gas Chromatograph 5890 series II (inlet 105°C, 30 m × 0.32 mm Omega-wax 320 capillary column; 30°C 5 minutes, 20°C/minute to 100°C, hold 15 minutes) and an FID detector (220°C) were used. The limit of reliable quantitation was approximately 5 μg/g.
Results and Discussion
Previous work has indicated that water is the preferred solution for extracting EO residues.8, 10 Yet the additional use of a nonpolar extraction solvent in testing medical devices is recommended.11,12 However, if the EO rapidly reacts with the components of the extraction solution or there is a significant dampening of the signal response, it may not be a reasonable solution to evaluate residual levels. To address this, a series of solutions was screened to compare simple signal response to EO from 0 to 50 μg/mL. Figure 1 shows the standard curves of EO in five different solutions. Of these, water clearly has the highest response. The RPMI with no dye and no FBS added was slightly higher than the other solutions (average 70% of the water peak signal). The remaining solutions were all around 55% of the water response.
The response of an EO analytical standard may differ from the response of EO extracted from a device because the EO analytical standard used in this study is available in methanol. The addition of even a small amount of a different solvent can change the properties of a solution (cosolvent effect). Therefore, the same amount of methanol used in the analytical standard solution was added to the extracts of the four devices and two materials. These devices and materials were extracted with water, cell culture media with 10% FBS and glutamine, and FBS to determine EO residual levels. Figure 2 shows the average peak area of residual EO in 35-mm film after extraction in water, media, or FBS. Zero percent, 1%, and 5% methanol was added to evaluate any potential cosolvent effect from the methanol. There is no significant difference with the increase of methanol up to 5%, but there is a lower response with the media and FBS with 5% methanol added. The media and FBS peak areas are approximately 60% and 45% lower than water, respectively. Figure 3 compares the EO residual level from these same 35-mm film samples after the peak area was extrapolated to determine the concentration using analytical standards prepared in the same solution. There is no significant difference between the extraction solutions, although the FBS extracted film had a lower response. To ensure all co-solvent effects were controlled for, sample extracts had 0%, 1%, or 5% methanol added.
Similar results were seen with two angiographic catheters (Figure 4); there was no significant difference in EO residual level when water, media, or FBS was used as the extraction solvent.
This was not always the case. Figure 5 shows the EO residual levels of a peripheral balloon dilation catheter extracted with water, media, or FBS. The device extracted with media and FBS show approximately 30% lower EO residual levels. However, water did not always yield the same or highest EO levels. In Figure 5, the electrophysiology catheter had a 20% higher EO level when extracted in media as compared to water. This may be due to the normal variability associated with extraction and analysis, where intralaboratory variability approaches 50% RSD.13
Various potential extraction solutions fortified with EO had up to a 50% difference in signal response between water, various cell culture media, and FBS. However, it is the residual EO values generated after extracting various devices and materials that is of real concern. After extracting various devices and materials with water, media, and FBS, there was no larger than a 30% difference in the EO residual levels. If one solution consistently generated higher EO residual levels, this would require further examination as to which solution gave the "correct" values. However, this was not the case. Water gives a higher signal than media or FBS, but no solution consistently gave a higher concentration value. Both the media and FBS had noise in the area where the EO peak elutes. Given the coeluting peaks when using media or FBS, and the higher cost of purchasing media or FBS, water is an adequate extraction solution to determine residual EO levels.
We would like to thank Paula Wren, Theresa Gaymon, and Racquel O'Neal-Felix at the National Institutes of Health for EO packaging and sterilization and HW Cyr and Gail Matson at FDA for their review and comments. From the Center for Device and Radiological Health, U.S. Food and Drug Administration, Silver Spring, MD. 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 FDA. Address correspondence and reprint requests to Dr. Lucas, Center for Device and Radiological Health, U.S. Food and Drug Administration, 10903 New Hampshire Ave., Life Sciences Building 64, Silver Spring, MD 20903 (e-mail: email@example.com).