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Residual Ethylene Oxide: Levels in Medical Grade Tubing and Effects on an In Vitro Biologic System R. G. MC GUNNIGLE, J. A. RENNER, S. J. ROMANO, and R. A. ABODEELY, JR. Eihicon, Inc., Somerville, New Jersey 08876 Summary The level of residue! ethylene oxide after sterilization was evaluated as a func tion of aeration time for three medical grade tubings. Toxicity resulting from residual ethylene oxide was determined in an in vitro tissue culture system uti lizing L-cells. The absorption and desorption of ethylene oxide from poty(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^J INTRODUCTION Ethylene oxide is a generally accepted sterilant for many plastic materials intended for medical applications. Its bactericidal effect i.s most probably due to aklylation, i.e., the replacement of an avail able hydrogen atom with a hydroxyethyl group. This reaction can inactivate many sulfhydryl, amino, or carboxyl groups of human protein molecules which may result in burns, hemolysis of blood and tissue necrosis. The potential toxicity of ethylene oxide is signifi cant since gas may be absorbed during the sterilization process and may remain in the materials for long periods of time.4 It is important that, prior to use, residual ethylene oxide in such materials be reduced to safe levels since its potential effects may be harmful to patients. Because of potential toxic reactions, Yasuda, Refojo and Stone* recommended that precautions be taken to minimize residual ethyl ene oxide levels, especially when ethylene oxide sterilized polymeric 273 (c) 1975 by John Wiley & Sons, Inc. V' 274 MC GUXXIC3LE ET AL. materials are used as surgical implants. Specifically, they noted that it would be good practice to include evacuation periods of at least 1 hr following sterilization before breaking the vacuum. * Hirose et al.1 reported that at least 3 and preferably 5 days should elapse before using ethylene oxide-sterilized plastic tubing for a clinical extracorporeal bypass. Roberts* recommended that surgical medical devices stand at least 5 days at room temperature or 8 hr at 120F before use. However, the numerous variables associated with ethylene oxide sterilization, e.g., gas concentration, temperature, relative humidity, material composition and packaging requirements, make it extremely difficult to delineate effective aeration conditions for all materials. In addition, generally accepted methods for routine chemical deter minations of residual ethylene oxide and its effects on biologic systems have not been firmly established though many groups art. actively working on these projects.7 This investigation was undertaken to investigate the removal of residual ethylene oxide from three types of medical tubing as a function of aeration time following sterilization. In addition, the effects of residual ethylene oxide on an in vitro biologic system are described. MATERIALS AND METHODS Tubing Materials Segments of poly(vinyl chloride) (Tygon, Norton plastics), silicone (Silastic, Dow Corning), and polyether-polyurethane (Biomer, Ethicon, Inc.) approximately 1 cm long and weighing 250 mg, were used for determination of residual ethylene oxide and in vitro biologic toxicity assays. Tubing was employed as received and had dimen sions of H in- id., K in- od. and Yi in. wall. Segments were exposed to ethylene oxide and aerated in 4 X 4 in. glassine bags. Ethylene Oxide Exposure and Aeration The tubing samples were exposed to ethylene oxide in a modified Cryotherm unit (Amsco) with & 46 1. capacity chamber. The condi tions of exposure were: 2200 mg/1 ethylene oxide (100% E.O. gas); 130F =b 5; <10% relative humidity; and 90 min holding time. r 21481002 **v ; UKSIDUAL ETHYLENE OXIDE 27. Following exposure, sanjples were aerated in the unit for various times up to 24 hr at 130*F 5 and 30 in. vacuum. Samples were removed from the unit, after appropriate aeration, for the simultane ous determination of residual ethylene oxide by the gas liquid chromatograph head space method, and of toxicity by an in vitro tissue culture method. Analysis of Ethylene Oxide Residue The head space method8 was used to determine residual ethylene oxide in the tubing samples. The method measures the amount of ethylene oxide gas volatilized into the gaseous head space of an en closed vial. Following exposure to ethylene -oxide and aeration, tubing samples (0.1 to 1 g) were placed in septum-sealed vials and heated for 20 min at 100C. To obtain peak height response, gaseous aliquots (100 mD from the head space were injected into a calibrated Hewlett-Packard 5750 gas chromatograph equipped with a 6 ft X in. S.S. Porapak R. column. Thereafter, the vial was purged with dry nitrogen, reheated and aliquots of gas were injected into the chromatograph as described. A third heating and aliquoting was usually required to extract all the ethylene oxide from poly(vinyl chloride) samples containing high levels (usually above 2000 ppm) of the gas. The amount of ethylene oxide in the sample was calculated using the sum of the peak heights from the two (or three) sample heatings and the calibration factor. In Vitro Tissue Culture A modified agar overlay method8'30 was used to determine the in vitro toxicity of the samples. Eagle's minimal essential medium (MEM) supplemented with 10% fetal calf serum was used for growth and maintenance of all cultures. All media contained penicillin (100 units/ml) and streptomycin (100 jjg/ml). Approximately 5 X 10* cells/ml of a continuous line of mouse L-cells (Clone 929) grown in Spinner MEM were placed in 60 mm plastic Petri dishes (Lux Scientific) and allowed to grow as monolayers in a. humidified atmosphere of 5% COj at 37C. When confluent, (usually 48 hr) the medium was decanted, the cells washed in phosphate-buffered saline (pH 7.2) and the test samples were placed directly on the monolayer. Three ml of previously prepared media composed of equal parts of 2X Eagle basal medium without phenol red and 2% Xobel -Mr 276 MC GUNNIGLE ET AL. agar adjusted to pH 7.2 and containing 0.01% neutral red as a vital stain, were added to the plates. After incubation for 24 hr in a 5% COj atmosphere at 37C, the plates were read for evidences of toxi city. Toxicity was characterized by relatively clear, colorless zones of dead cells which had lost the vital stain surrounding the sample. Cytotoxic effects were confirmed by microscopic examination of the cells surrounding the test sample. EXPERIMENTAL RESULTS Residual Ethylene Oxide in Poly(vinyl Chloride) Tubing The effects of aeration at 130T at 30 in. vacuum on the reduction of residual ethylene oxide in the poly(vinyl chloride) tubing samples as well as the corresponding in vitro tissue culture reactions are depicted in Table I. The initial level of residual ethylene oxide, immediately following exposure and prior to aeration, was greater than 17,000 ppm. At this level, the tubing was highly toxic to the L-cells, causing complete destruction of all cells on the Petri dish. A similar, highly toxic reaction was noted in samples aerated for 1 hr which contained residual ethylene oxide levels of 10,000 ppm. TABLE I Effects of Aeration on Residual Ethylene Oxide and In Vitro Biologic Response of Polyvinyl Chloride Tubing* Aeration (hr) Residual E.O. (ppm) Lrcetl toxic cone (mm) 0 l 2 3 4 5 6 6.5 7 24 17,200 10,000 5,340 3,570 3,160 2,220 1,510 900 420 34 >25 >25 8 3.5 3.5 3.1 2.8 0 0 0 Tubing segments were exposed to 2200 mg/1 ethylene oxide (100% E.O.) at 130F =t 5, < 10% relative humidity for 90 min. Aeration was accomplished in the unit for the above times at 130F 5 with 30 in. vacuum. BFG14284 21481004  x*-% RESIDUAL ETHYLENE OXIDE 277 \ Aeration from 2 to 5 resulted in a decrease in the residual ethylene oxide level fronr 5,340 ppm to 2,220 and a concomitant decrease in the in vitro tissue culture toxic zone from S mm to 3.1 mm. After 6 hr aeration, the residual ethylene oxide was reduced to approx imately 1500 ppm, with a corresponding reduction in the size of the tissue culture toxic zone to 2.8 mm. Approximately 900 ppm residual ethylene oxide was found in samples aerated for 6V hr, and 400 ppm at 7 hr. These samples showed no macroscopic or microscopic evidences of toxicity to the L-cells. Following 24 hr aeration, the level of residual ethylene oxide was 34 ppm, with no corresponding toxicity to the tissue culture cells. Control tubing segments, not exposed to ethylene oxide were non-toxic to the tissue culture cells. The in vitro tissue culture reactions corresponding to the above levels of residual ethylene oxide following aeration for various times are illustrated in Figures 1-4. The magnitude of the toxic zone indicates the relative toxicity associated with the amounts of residual ethylene oxide in the tubing. Unaerated samples and those aerated for 1. hr resulted in complete destruction of all cells on the Petri dish, Fig. 1. Macroscopic view of in vitro tissue culture response of I/-cells to poly(vinyl chloride) tubing segments following exposure to ethylene oxide and aeration as described in text. Tubings were embedded in the agar in contact with the L-cells and incubated for 24 hr at 37*C in 5% carbon dioxide. Tubing aerated for 2 hr containg 5,340 ppm residual ethylene oxide. Sample is sur rounded by toxic zone (clear area) containing dead cells caused by residual ethylene oxide. Remaining area is non-toxir and consists of viable cells which have retained the neutral red vita! stain. *%. 278 f MC CUXXICLE KT AL. Fig. 2. Genera! information same as for Fig. 1, but tubing has been aerated for 3 hr containing 3,570 ppm residual ethylene oxide. Xote toxic zone immedi ately surrounding sample. indicating extreme toxicity at these levels. In contrast, samples aerated from 2 to 6 hr did not cause destruction of all cells, some of which were surrounded by toxic zones of various sizes (Figs. 1-3). Figure 4 shows the absence of a toxic zone in samples aerated for over 6^4 hr and containing 900 ppm or less residual ethylene oxide. The figure also reveals the reaction elicited by the unexposed control tubing. Fig. 3. General information same as for Fig. 1, but tubing has been aerated for 6 hr containing 1,510 ppm residual ethylene oxide. Note very slight toxic zone surrounding sample. 24481006 RESIDUAL ETHYLENE OXIDE 279 VJtg -w pxvr Fig. 4. General information same as for Fig. 1, but tubing has been aerated for hr containing 900 ppm residua) ethylene oxide. Note absence of clear zone surrounding sample indicating absence of toxicity. Photograph is atso representative of response elicited by control tubing (i.e. not exposed to ethylene oxide.) Photomicrographs of the cellular areas surrounding the samples are seen in Figures 5 and 6. Figure 5 is representative of the cellular response to samples containing 1,500 ppm or more residual ethylene oxide. The neutral red vital stain originally present in the cell has been lost; the cells exhibit rounded morphology and are dead. In contrast, the cells surrounding the unexposed and exposed samples having 900 ppm or less residual ethylene oxide, seen in Figure 6, have retained the neutral red vital stain, exhibit characteristic fibroblast-like morphology and are viable. Retention of Residual Ethylene Oxide in Silicone, Poly(vinyl chloride), and Polyether-Polyurethane Tubing The results of comparative studies on the retention of residual ethylene oxide in silicone, poly(vinyl chloride), and polyetherpolyurethane tubings are presented in Table II and Figure 7. These indicate that the initial absorption of ethylene oxide is essentially similar for both the poly(vinyl chloride) and polyether-polyurethane tubing. The levels of ethylene oxide following exposure and prior to aeration were between 15,000 and 16,000 ppm in each material. In contrast, silicone tubing initially retained about 2100 ppm ethylene oxide. This is equivalent to approximately 85% absorption of BFG14287 280 MC GUNXICLE ET AL. u *// i r. * V^..Vrffilj & ssh <$ ^ .V.' --* ,' -^--vA 5^5^?': ft- ..-- .^ "J* $ * + '&&*? Fig. 5. Photomicrograph (X400) of L-cells surrounding poly(vinyl chloride) tubing (shaded area lower left) containing 1,500 ppm or more residual ethylene oxide. Note that the cells have lost the neutral red vital stain, exhibit pre dominantly rounded morphology and are desd indicating the sample is toxic to the cell. gas by the silicone compared to poly(vinyl chloride) and polyetherpolyurethane materials. The residual ethylene oxide in the poly(vinyl chloride) and polyether-polyurethane tubing was reduced to less than 150 ppm after aeration for 8 hr. This level revealed no macroscopic or microscopic TABLE II Effect of Aeration on Elution of Residual Ethylene Oxide from Silicone, l'oly(vinvl Chloride) and Polyether-Polyurethane Tubings* Residua] Ethylene Oxide (ppm) Aeration (hr) Silicone PolyetherPoly(vinyl chloride) polyurethane 0 2120 15,500 15,300 2 .50 4,200 4,900 5 7 1,000 1,100 8 1.5 142 131 24 3 131 111 Tubing segments were exposed to 2200 mg/I ethylene oxide (100% E.O.) ut 130F 5, < 10% relative humidity for 00 min. Aeration was accomplished in the unit for the above times at 130F d: o with 30 in. vacuum. N 1481008 BFG14288 ide) Fig. 6. Photomicrograph (X400) of L-cells surrounding poiy(vinyl chloride) lette tubing (shaded area lower left) containing 800 ppm or less residual ethylene pre- oxide. Note that the cells retain the neutral red vital stain, exhibit character jxic istic fibroblast-like morphology and are viable indicating the sample is non-toxic to the cells. This reaction is typical of the unexposed control tubing. fr evidence of toxicity in the in vitro tissue culture assay. In contrast, the residual ethylene oxide in the silicone tubing was reduced to SO ppm following 2 hr aeration and less than 2 ppm after 8 hr. The eer difference in aeration time necessary to reduce the residual ethylene pic oxide to safe levels ostensibly reflects the greater amount initially present in the poly(vinyl chloride) and polyether-polyurethane materials. lv- DISCUSSION One of the problems associated with ethylene oxide sterilization is potential toxicity of the residual ethylene oxide. Plastic, rubber, leather, and porous materials of all types have been shown to absorb varying amounts of the gas during exposure.7'11 'lI Stetson and Guess1*, O'Leary and Guess*, Guess11, Matsumoto et al.4 and Anderson7 have investigated these parameters in some detail. Their reports indicate that, in general, absorption depends not only on the composition of the material but also on the specific conditions .. H ;e<| employed during the sterilization cycle. Desorption is also depend ent on the composition of the material, packaging, and aeration conditions, e.g. ambient, high temperature or vacuum aeration. QO y^ O o CD BFG14289 *V ' 282 MC GUNNIGLE ET AL. Fig. 7. Ethylene oxide content in plastic tubings vs. aeration time. Tubing segments (1 cm and 250 mg) were exposed to 2200 mg/i ethylene oxide (100% E.O.) at 130F 5, < 10% relatively humidity for 90 min. Aeration was accomplished in the unit at 130CF 5 with 30 in. vacuum. Po!y(vinyl chloride) (), polyether-polyurethane (D, and silicone (A). It is extremely important that the parameters for ethylene oxide sterilization, subsequent aeration, and the effects of residual ethylene oxide on biologic systems be defined for each type of material or product sterilized. The time required to reduce the residual ethylene oxide to safe levels depends on the above mentioned factors. The results presented in this paper indicate that, under the conditions employed, silicone tubing absorbed approximately 85% less ethylene oxide than either poly(vinyl chloride) or polyether-polyurethane tubing. In addition, the aeration time required to reduce the ethylene oxide to safe levels, as detected by the in vitro tissue culture method, reflected the amount of gas initially absorbed in the material. 21481010 , , RESIDUAL ETHYLENE OXIDE 2S3 Silicone tubing required Ihr while poly(vinyl chloride) tmd polyethcr- polvurethane tubing required 7 to S hr. The use of the head space method in conjunction with in vitro tissue culture techniques provides an efficient, rapid (less than 24 hr) and inexpensive detection and assay system. The procedure of embedding the sample material in the agar overlay in direct contact with the tissue cells showed that residual ethylene oxide levels of approximately 900 ppm or more were toxic, while samples containing less were non-toxic. The authors wish to thank Mrs. Luzianna Palmer and Mr. John Hughes for their expert technical assistance. References 1. T. Hirose, K. Goldstein, and C. Bailey, J. Thoroc. Cardioo. Surg., 45, 245 (1963). 2. R. K. O'Leary and W. L. Guess, J. Pharm. Set., 57, 12 (1968). 3. R. E. Roberts and L. Rendell-Baker, Med. Surg. Rec., 10-14, Fourth quart., (1969). 4. I. Matsumoto, R. M. Hardaway, K. C. Pani, C. M. Sater, 1). E. Bartak, and P. M. Margetis, Arch. Surg., 96, 464 (1968). 5. H. Yasudo, H. F. Refojo, and W. Stone. Papers presented at the meeting of the American Chemical Society, Division of Organic Coatings and Plastic Chemistry 209, Chicago, 1964. 6. R. E. Roberta, Med. Surg. Rev., 3, Fourth Quart. (1968). 7. S. R. Anderson, Bull, of Parenteral Drug. Assoc., 27, 249 (1973). S. S. Romano, J. L. Renner, and P. M. Leitner, Anal. Chem., 45, 2347 (1973). 9. S. A. Rosenbluth, G. R. Weddington, W. L. Guess, and J. Autian, J. Pharm. Sci., 54, 156 (1965). 10. J- A. Taylor, R. A. Abodeely, and It. L. Fuson, Trans. Anter. Soc. A riif. Jnt. Organs, 19, 175 (1973). 11. W. L. Guess, Bull. Parenteral Drug. Assoc., 26, .58 (1972). 12. T. I>. McDonald, K. Hasten, R. Harvey, S. Gregg, A. R. Borgmann, and T. Murchisan, Bull. Parenteral Drug. Assoc., 27, 4, 153 (1973). 13. J- B. Stetson W. L. Guess, Int. Anesthesial Clin., 8, 829 (1970). Received July 1, 1974