Ethylene oxide was tested on groups of rats for either 4-hour or 1-hour inhalation exposure, followed by 14 days of observation. Groups of five Sprague-Dawley rats/sex were exposed, and clinical signs and mortality were recorded. Clinical signs noted included irregular breathing, absence of certain reflexes, and tremors. Rats that died had moderate to severe pulmonary congestion. The calculated LC50 values, reported as ppm by volume (with 95% confidence limits), were as follows. 4-hour LC50 values were 1972 (1887 to 2061) ppm for males; 1537 (1391 to 1698) ppm for females; 1741 (1655 to 1831) ppm for the combined sexes. The 1-hour LC50 values were 5748 (5276 to 6262) ppm for males; 4439 (4034 to 4884) ppm for females; 5029 (4634 to 5459) ppm for the combined sexes.
Ethylene oxide (EO, CAS no. 75-21-8) is a colorless liquid, boiling at 10.7°C (760 mmHg) and has a characteristic ether-like odor. It is extremely flammable and can form an explosive mixture with air. It is an important industrial chemical intermediate and has limited use as a low-temperature sterilant, mainly for use on medical equipment and spices, because of its very high reactivity characteristic. EO has been classified as a Toxic Inhalation Hazard chemical by the US Department of Transportation [
There are several studies on the 4-hour acute effects from EO exposure to rats published more than 50 years ago. Justification for selection of the Threshold Limit Value, originally set at 100 ppm, from the early years (1946–1956) of ACGIH efforts [
Another acute inhalation study was reported by Carpenter et al. [
It is also noted that RTECS [
Hence, a more robust 4-hour LC50 study in rats was conducted because of the discrepancies between the older studies. In addition, since a 1-hour LC50 value in rats is needed for certain risk assessments (e.g., DOT, [
Stainless-steel gas cylinder partially filled with liquid EO (CAS no. 75-21-8) was obtained from Union Carbide Corporation. Results of compositional analysis indicated that the substance was 99.9% pure EO.
Stainless-steel gas cylinders containing different concentrations of EO in air (ranging from 4000 to 7000 ppm EO) were obtained from Union Carbide Corporation as GC certified levels of EO.
Young adult male and female Sprague-Dawley albino rats (approximately 6 weeks of age) were obtained from Harlan Sprague-Dawley, Inc., Indianapolis, Ind, USA. Animals were acclimated to facility environment approximately 1 to 2 weeks before treatment and were on a 12-hour light/dark cycle. The approximate group weight range on the first day of exposure for the 4-hour was 220–280 kg for males and 180–195 kg for females; for the 1-hour was 250–285 kg for males and 185–200 kg for females. Food (pelleted Pro Lab RMH #3000, Agway, Inc.) and municipal water were provided
The cylinder of liquid EO was maintained at approximately 35°C in a water bath. The resulting headspace of EO vapor was regulated through stainless-steel tubing and delivered through a calibrated flowmeter into a mixing chamber with filtered-room air before being carried into the inhalation chamber (under negative pressure).
The EO vapor (certified concentration) was regulated from the cylinder through stainless steel tubing. The vapor was then metered through a calibrated flowmeter into the inhalation chamber, and when necessary, the EO was diluted with filtered-room air before delivery into the inhalation chamber (under positive pressure).
The rats were individually housed in wire-mesh cages and exposed (whole body) in a 1300-liter glass and stainless steel chamber (Rochester-type chamber). The chamber shape was rectangular with a pyramidal top and bottom with the EO vapor mixing with filtered air on the inlet side of the chamber. The calibrated chamber airflow rate was approximately 300 L/min.
The 1-hour exposures were conducted in a 120-liter Plexiglas and stainless steel chamber that was rectangular in shape. Since the exposure chamber was under a positive pressure, this 120-liter chamber was placed in a negative pressure containment system for safety. Because of the short exposure duration, the test vapor atmosphere was generated in the chamber before placement of the animals into the chamber. After maintaining the desired EO target exposure concentration, the animals, 5/sex in a wire-mesh cage (whole body exposure), were quickly inserted into the chamber by a sliding draw mechanism, and at the end of one hour, they were quickly removed. The calibrated chamber airflow rate was approximately 30 L/min.
A Perkin-Elmer Model 8500 or 3920B gas chromatograph (GC) equipped with a flame ionization detector was used to analyze the exposure chamber atmosphere. Calibration of the GC was achieved by injecting gas standards, which were prepared by volumetric dilution of certified EO standards, provided by Union Carbide Corporation. Each exposure chamber atmosphere was analyzed for EO approximately every 15 minutes (1-hour study) or every 30 minutes (4-hour study). Ethylene oxide concentration is reported as ppm by volume (ppm).
No control (air-only) exposures were performed. Body weight and health status were assessed before rats were randomly assigned into groups. Each group contained either 5 male or 5 female rats, unless it was determined that one sex did not have to be exposed to a different concentration based on mortality data from a previous exposure. The chamber temperature and relative humidity were recorded during exposure. Animals were observed for signs of adverse effects during and following the exposure. During the exposure, observations were recorded for all animals as a group, approximately every 10 minutes. During the 14 days following the exposure, all animals were observed at least once daily (typically morning and evening), and any overt clinical signs were recorded. The animals were weighed prior to exposure and on postexposure days 7 and 14. The change in body weight was calculated by subtracting the preexposure value from each successive weight. A complete necropsy was performed on all animals. The survivors were lightly anesthetized with methoxyflurane and killed by exsanguination. The lungs were weighed at necropsy.
Lungs were examined histologically from two exposure groups (4-hour study only) in which most of the animals survived (1850 ppm and 1443 ppm, male and female rats, resp.) and from two exposure groups from which most rats died following exposure (2182 ppm and 1850 ppm, male and female rats, resp.). At necropsy, the lungs were fixed by inflating with 10% neutral buffered formalin. The lungs were then embedded in paraffin, sectioned at 5-6 microns, and examined microscopically after hematoxylin and eosin staining.
The mean and standard deviation of the body weights, body weight changes, lung weights (4-hour study only), and exposure concentrations were calculated. No statistical comparisons were made. The LC50 values were determined by the moving average method of Thompson [
A number of atmospheric samples were taken to show that the controls on the vapor generation system were sufficient and resulted in a relatively stable exposure concentration for each exposure group. For the 4-hour study, the mean (±SD) analytical concentrations of EO (
Clinical signs noted included ocular and nasal irritation, irregular breathing, absence of certain reflexes, ataxia, and tremors. No rats died during any of the 4-hour exposure periods. One male rat died within 1 hour following exposure to the highest level (2182 ppm). All remaining deaths occurred within three days following exposure. For all groups, no clinical signs were observed in survivors after postexposure day 5. Tables
Clinical observations, mortality rates, and day of death for male rats following 4-hour exposure to EO.
Exposure group (ppm) | Clinical signs | Mortality | Number found dead | ||||
During exposure or same day following exposure | During five days of postexposure period | Number dead/total | 0a | 1 | 2 | 3 | |
2182 | Periocular/perinasal/perioral wetness, gasping, audible respiration, hypoactivity | Periocular/perinasal/perioral encrustation, unkempt fur, decreased respiration rate, hypoactivity | 4/5 | 1b | 2 | 1 | 0 |
2026 | Periocular/perinasal/perioral wetness, gasping, audible respiration, hypoactivity, absence tail/toe pinch reflex | Periocular/perinasal/perioral encrustation, unkempt fur, gasping, audible respiration, decreased respiration rate, hypoactivity, tremors, | 4/5 | 0 | 3 | 1 | 0 |
1850 | Periocular/perinasal wetness, gasping, audible respiration, hypoactivity | Perioral/perinasal encrustation, unkempt fur, audible respiration, decreased respiration rate, hypoactivity | 0/5 | 0 | 0 | 0 | 0 |
aDay 0 is exposure day. Days 1, 2, and 3 are days following exposure day.
bNumber of animals found dead on a particular day.
Clinical observations, mortality rates, and day of death for female rats following 4-hour exposure to EO.
Exposure group (ppm) | Clinical signs | Mortality | Number found dead | ||||
During exposure or same day following exposure | During five days of postexposure period | Number dead/total | 0a | 1 | 2 | 3 | |
1850 | Periocular/perinasal wetness, gasping, audible respiration, hypoactivity | Periocular/perinasal encrustation, unkempt fur, audible respiration, decreased respiration rate, hypoactivity | 5/5 | 0b | 3 | 2 | 0 |
1637 | Periocular/perinasal/perioral wetness, gasping, audible respiration, hypoactivity, absence tail/toe pinch reflex | Perinasal/perioral encrustation, unkempt fur, audible respiration, decreased respiration rate, hypoactivity, tremors | 4/5 | 0 | 3 | 0 | 1 |
1443 | Periocular/perinasal/perioral wetness, gasping, audible respiration, hypoactivity, tremors | Perinasal encrustation | 1/5 | 0 | 1 | 0 | 0 |
aDay 0 is exposure day. Days 1, 2, and 3 are days following exposure day.
bNumber of animals found dead on a particular day.
Clinical signs noted included ocular and nasal irritation, irregular breathing, absence of certain reflexes, ataxia, and tremors. Group observations during exposure for all groups included hyperactivity for approximately the first 10 minutes of exposure followed by hypoactivity. No rats died during the 1-hour exposure period. All remaining deaths occurred within two days following exposure. For all survivors, no clinical signs were observed after postexposure day 2 (males) or day 3 (females). Tables
Clinical observations, mortality rates, and day of death for male rats following 1-hour exposure to EO.
Exposure group (ppm) | Clinical signs | Mortality | Number found dead | |||
During exposure | During two days of postexposure period | Number dead/total | 0a | 1 | 2 | |
6161 ppm | Absent startle reflex, gasping | Unkempt fur, decreased respiration rate, hypoactivity, ataxia, tremors | 4/5 | 0b | 3 | 1 |
5546 ppm | Absent startle reflex | Unkempt fur, decreased respiration rate, hypoactivity, ataxia, tremors | 1/5 | 0 | 0 | 1 |
4827 ppm | Absent startle reflex | Perioral/perinasal encrustation | 0/5 | 0 | 0 | 0 |
aDay 0 is exposure day. Days 1 and 2 are days following exposure day.
bNumber of animals found dead on a particular day.
Clinical observations, mortality rates, and day of death for female rats following 1-hour exposure to EO.
Exposure group (ppm) | Clinical signs | Mortality | Number found dead | |||
During exposure | During three days of postexposure period | Number dead/total | 0a | 1 | 2 | |
4827 ppm | Absent startle reflex | Perinasal wetness, pericular/perioral/perinasal encrustation, unkempt fur, decreased respiration rate, hypoactivity | 5/5 | 0b | 1 | 2 |
4202 ppm | Periocular/perinasal encrustation, unkempt fur, decreased respiration rate, hypoactivity, ataxia, tremors | 1/5 | 0 | 4 | 1 | |
3966 ppm | Unkempt fur, decreased respiration rate, hypoactivity, ataxia, tremors | 2/5 | 0 | 0 | 1 |
aDay 0 is exposure day. Days 1 and 2 are days following exposure day.
bNumber of animals found dead on a particular day.
All body weight changes for surviving animals from day 0, the exposure day, showed an increase at postexposure day 7 and day 14. Although no statistics were conducted, the rate of body weight gain was greater in groups that had lower mortality incidence.
No control group was used in this study. The principal gross findings observed in animals that died included diffuse or multifocal discoloration of the lungs and hyperinflation of the lungs. Clear fluid in the thoracic cavity was recorded in one male and one female that died in the highest exposure groups in the 1-hour study. Focal or multifocal color change of the lungs was the only significant gross finding observed in animals euthanized 14 days following the exposure. This finding was observed sporadically. Additional gross findings observed in rats that died or were sacrificed were attributed to autolysis or regarded as incidental findings. Based on laboratory historical findings, all lung weights (determined only in the 4-hour study animals) of surviving rats were in the normal range. Accurate lung weights could not be obtained for animals that died prior to scheduled terminal sacrifice (14 days postexposure).
No control group was used in this study. Based on laboratory historical findings, the lesions noted are attributed to treatment. Lungs from males and females were examined histologically from the highest and the lowest exposure groups, including animals that did not survive until 14 days postexposure. Treatment-related microscopic lesions were seen in all the lungs of males of the 2182 ppm group and all the females of the 1850 ppm group that died, all by day 2 postexposure (Table
Lung histopathology of animals that died following 4-hour exposures to EO.
Microscopic diagnosis | Male | Female | ||
2182 ppm | 1850 ppm | 1850 ppm | 1443 ppm | |
Number found dead/sacrificed moribund | 4 | 0 | 5 | 1 |
Lungs | ||||
Congestion, moderate, marked, or severe | 4/4 | 0 | 5/5 | 1/1 |
Pulmonary edema, minimal or moderate | 2/4 | 0 | 0/5 | 0/1 |
Emphysema, moderate | 1/4 | 0 | 0/5 | 0/1 |
Alveolar histiocytosis, mild | 1/4 | 0 | 0/5 | 0/1 |
Hemorrhage, mild | 3/4 | 0 | 3/5 | 0/1 |
Pneumonitis, interstitial, mild | 1/4 | 0 | 2/5 | 0/1 |
The LC50 calculations for both the 4-hour and 1-hour studies are given in Table
LC50 calculations reported in ppm by volume and converted to mg/m3.
Unit of measurement | 4-hour LC50 | 1-hour LC50 | Ratio 1-hour/4-hour | |
---|---|---|---|---|
Males | ppm | 1972 (1887–2061) | 5748 (5276–6262) | 2.9 |
mg/m3 | 3550 (3397–3710) | 10346 (9497–11272) | 2.9 | |
Females | ppm | 1537 (1391–1698) | 4439 (4034–4884) | 2.9 |
mg/m3 | 2767 (2504–3056) | 7990 (7261–8791) | 2.9 | |
Combined sexes | ppm | 1741 (1655–1831) | 5029 (4634–5459) | 2.9 |
mg/m3 | 3134 (2979–3296) | 9052 (8341–9826) | 2.9 |
High concentrations of EO do cause death in acutely exposed rats. In the published history of EO use and accidents in the United States, no original citation has been found that reports a human fatality from acute inhalation exposure as would be expected for a toxic inhalation hazardous material. In agreement with this nonevent is a recent review by the National Research Council [
From the clinical observation and pulmonary histology examination in the rat 4-hour study, it is not known if the resulting mortality was the result of a severe systemic toxicity or due to some mechanism which interferes with normal respiratory function. In both the 1-hour and 4-hour studies, clinical signs of ataxia, tremors, absence of the startle reflex, absence of the tail/toe pinch reflex, and decreased respiration rate were noted, and all of these could have a neurologic effect from EO exposure as a component. In humans, reports of dizziness and repeated vomiting also support a potential neuropharmacologic action of EO [
Moderate to severe pulmonary congestion was the most significant finding in the animals that died following acute exposure to EO. Caution must be taken in the interpretation of the other lung lesions identified in animals that were found dead. For example, autolysis can mimic pulmonary edema, and diapedesis of red blood cells from congested capillaries can mimic mild hemorrhage, both of which were recorded in this study. Microscopic findings were not significant for the one male rat that survived exposure to the highest exposure level, with the exception of mild multifocal emphysema. Emphysema in this animal may have been due to dyspnea at the time of exposure. In addition, some of these could be agonal changes. These pulmonary findings are similar to those reported by Jacobson et al. [
Past practice has been to conservatively estimate the 1-hour LC50, in the absence of animal data, by multiplying a 4-hour LC50 value by 2 [
These two observations are related, since when two exposure paradigms (concentration
Parts of this work were supported by the Industrial Chemicals and Linde Divisions of Union Carbide Chemicals and the Balchem Corporation. The authors gratefully acknowledge Irvin Pritts, Ph.D., for his assistance in chemical and chamber atmosphere analyses, and P. E. Losco, VMD for her evaluation of the lung tissues; the authors are grateful to N. P. Moore, Ph.D., for careful review and valuable comments on the paper.