Ethylene oxide

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

toxicokinetics

Qualifier:

no guideline followed

Principles of method if other than guideline:

Effects of acute EO exposure on GSH content of ten tissues in adult rats and mice.

GLP compliance:

not specified

Radiolabelling:

not specified

Species:

other: rats and mice

Strain:

other: Fischer-344 and Swiss-Webster

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: Male rats (180 - 210 g) and male mice (28 - 32 g)
- Diet: ad libitum
- Water: ad libitum

Route of administration:

inhalation

Details on exposure:

Air flow through the system was approx. 9l/min. Air was sampled from the chamber at 15-min intervals and analyzed for EO concentration by gas chromatography. Measured levels did not deviate from target levels by mor the 10%. For each experiment, similar numbers of control animals were exposed to breathing quality air under identical conditions.
EO-exposed mice and 4 controls were sacrificed at each time point.

Duration and frequency of treatment / exposure:

4 hours

Dose / conc.:

100 ppm

Remarks:

mice/rat

Dose / conc.:

400 ppm

Remarks:

mice

Dose / conc.:

900 ppm

Remarks:

mice

Dose / conc.:

600 ppm

Remarks:

rat

Dose / conc.:

1 200 ppm

Remarks:

rat

No. of animals per sex per dose / concentration:

Groups of 4 rats or 4 - 20 mice

Control animals:

yes

Details on study design:

Immediately upon termination of EO exposure or 24 hours after exposure, rats and mice were sacrificed. Animals were anesthetized with methoxyflurane; the abdomen was opened and the animals were exsanguinated via the abdominal aorta or vena cava. Selected tissues were rapidly removed, weighed and immediately frozen in liquid nitrogen. The frozen tissue was placed in a vial on dry ice until all animals in a particular study were sacrificed. Blood removed during exsanguination was also frozen in liquid nitrogen and held on dry ice. When all animals were sacrificed, tissue samples were sequentially thawed and homogenized in two volumes of 0.1 M phosphate - 0.005 M EDTA buffer (pH=8.0) with a Polytron homogenizer.
Appropriate aliquots of tissue homogenate were added to 25% metaphosphoric acid, mixed, and centrifuged at 100,000g for 30 min. Reduced and oxidized glutathione (GSH and GSSG) contents of the clear supernatant were analyzed by the spectrofluorometric method. Protein content of each homogenate was measured by the Lowry method.
Since more mice than rats could be exposed in the inhalation desiccator at any one time, mouse studies were extended to examine the effects of different E0 exposure levels and also a wider range of sacrifice times on tissue GSH levels. Mice were sacrificed immediately after exposure or 6, 12, 24 or 48 hours after exposure. In addition, a group of 4 mice was given an i.p. injection of 0.6 ml/kg of diethylmaleate (DEM) in corn oil and sacrificed 1, 6, 12, 24 or 48 hours later. Tissue GSH levels after DEM are reported to facilitate comparison of GSH depletion caused by E0 exposure with depletion caused by DEM, a chemical known to produce a rapid, transient decrease in tissue GSH. Four E0-exposed mice and four controls were sacrificed at each time point.

Statistics:

The homogeneity of variances for glutathione levels in various treatment groups was determined by use of Bartlett's test. If the variances were homogeneous, F values for analysis of variance were determined. When F was significant, Duncan's multiple range test was used to denote intergroup differences. When variances were heterogeneous, the groups were compared, in pairs by the F test. When p < 0.05 for the F test, Cochran's t test was used to denote the significance of difference between means. When p < 0.05 for the F test, Student's t test was used to compare means.

Type:

metabolism

Results:

Immediately after exposure, GSH levels were significantly decreased in all tissues examined.

Details on absorption:

not determined

Details on distribution in tissues:

Distribution was confirmed in liver, lung, stomach, testis, bone marrow, blood, kidney, heart, brain, spleen.

Details on excretion:

Urinary excretion

Metabolites identified:

not measured

Details on metabolites:

not determined

Bioaccessibility (or Bioavailability) testing results:

GSH levels were significantly decreased in all tissues examined, with the exception of blood. The most significantly affected tissues (and the % GSH depletion) were: liver (82%), lung (72%), stomach (72%) and testis (63%). GSH depletion in other tissues ranged from approximately 20-50%.

Effects of EO (1200 ppm) on tissue GSH levels in the RAT:

In initial experiments, groups of 4 male rats were exposed to EO at a chamber level of 1200 ppm for 4 hours. The only significant differences in relative organ weights between EO exposed rats and chamber controls was a 17% increase in stomach weight immediately after exposure and an 11% increase in brain weight and a 30% decrease in spleen weight 24 hours after exposure. No significant differences in tissue protein levels were observed. GSH levels were significantly decreased in all tissues examined, with the exception of blood. The most significantly affected tissues (and the % GSH depletion) were: liver (82%), lung (72%), stomach (72%) and testis (63%). GSH depletion in other tissues ranged from approximately 20-50%. Twenty-four hours after exposure, GSH levels were still depressed in bone marrow (33%) and testis (35%). However, in all other tissues, GSH levels had returned to control values or had "rebounded" to levels slightly above control. The tissue concentrations of oxidized glutathione (GSSG) were also measured (data not shown). Levels of GSSG were either unaffected or decreased (lung, testis and liver) after EO exposure. Oxidized glutathione levels never increased in EO treated rats as would occur if GSH "depletion" was due to oxidation of GSH to GSSG.

Dose-response relationships in the RAT:

In order to determine the influence of different EO exposure levels on tissue glutathione concentrations, a second series of experiments were done. Rats were exposed to 100, 600 or 1200 PPm E0 for 4 hours and GSH levels were measured in selected tissues immediately after exposure. GSH levels were compared to control rats and presented as a percent of respective control. At 100 ppm EO, GSH levels were significantly depressed by approximately 20% in lung, testis and liver. At 600 ppm and 1200 ppm, GSH in all tissues, except blood, was depleted. Although GSH depletion in all tissues was dose dependent, the relationship was not linear (i.e., there was a much steeper slope between 100-600 ppm than between 600-1200 ppm).

Effects of EO (900 ppm) of tissue GSH Levels in the MOUSE:

Four male Swiss-Webster mice per group were exposed to EO (900 ppm) or air in the inhalation desiccator. The only significant differences in relative organ weights between E0 exposed mice and controls was a 39% decrease in spleen weight in E0 exposed animals 24 hours after exposure. No significant differences in tissue protein levels were observed. In all tissues, except kidney, GSH levels were significantly depleted immediately after termination of exposure. Consistent with the rat data, the most significantly affected tissues in mice (and the % GSH depletion) were: lung (86%), liver (85%) and stomach (69%). Unlike the rat, mouse testicular GSH levels were less affected (39% depletion) while GSH was depleted to a greater extent in the heart (69% depletion) and blood (71% depletion). Twenty-four hours after exposure, GSH levels were still below control levels in the blood and testis while levels rebounded above controls in the lung. Oxidized glutathione (GSSG) levels were unaffected in the kidney, stomach and spleen of E0 exposed mice immediately after exposure while depletion occurred in other tissues (ranging from 29% in the brain to 86% in the lung).

Dose-response and time-response relationships in the MOUSE:

Each panel shows the effect of an injection of the well known GSH-depleting agent DEM. For all tissues, the most significant GSH depletion was evident immediately after E0 exposure. By 6 hours, recovery had begun, or was complete, in each tissue. Immediately after E0 exposure, a dose-response relationship similar to that in rats was evident; the extent of depletion between 100-450 ppm was greater than between 450-900 ppm (except in the testis). E0 (450 ppm and 900 ppm) and DEM caused an immediate 60-70% decrease in GSH. By six hours, levels had rebounded above control in DEM-treated mice and returned to control in E0 (450 ppm) exposed mice. Levels remained this way for 48 hours in both groups. In contrast, mice exposed to E0 (900 ppm) had significantly depressed GSH levels for 12 hours, levels near control at 24 hours and levels slightly above control by 48 hours. E0 (100 ppm) had no effect on stomach GSH at any time point. In the testis, E0 (100 ppm and 450 ppm) was virtually without effect on tissue GSH levels. E0 (900 ppm) and DEM caused immediate GSH depletion of approximately 40%. Recovery was slow in both cases, levels not returning to control until 48 hr after DEM injection or E0 exposure (900 ppm). In the lung, 100 ppm or 450 ppm exposure to E0 caused immediate depression of GSH by approximately 20% and 60% respectively. At these exposure levels, GSH returned to control values by 6 hr and remained at control levels until 48 hr. E0 (900 ppm) produced an immediate 85% GSH depletion. Levels were still depressed by 50% after 6 hours and returned to control by 12 hr. DEM also produced an immediate depletion of 70%, however, levels returned to control at 6 and 12 hours and rebounded above control by 24 and 48 hr. In the liver, E0 (100 ppm) produced an immediate 20% depletion of GSH. Levels rebounded above control (20%) by 6 hr and returned to control at all other time points. E0 (450 ppm) and DEM both produced an immediate 60% depletion of GSH with levels not being significantly different from control thereafter. With 900 ppm E0 exposure, GSH depletion was evident for 24 hours (50-80%). Levels returned to control values by 48 hr after exposure. Although E0 had no effect on blood GSH levels in the rat, 450 ppm or 900 ppm produced an immediate and long lasting decrease in blood GSH in the mouse. Levels returned to control by 48 hr after 450 ppm, but were still depressed 48 hr after 900 ppm. DEM depleted blood GSH in a manner similar to 450 ppm of EO. EO (100 ppm) was without effect on mouse blood GSH levels.

Conclusions:

The results indicate a marked species difference between rats and mice regarding the effects of EO exposure on blood GSH levels which may have important toxicological implications.

Reference 2

Endpoint:

basic toxicokinetics, other

Remarks:

in vivo and in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Objective of study:

excretion

metabolism

toxicokinetics

Qualifier:

no guideline followed

Principles of method if other than guideline:

Defining the kinetics of removal of ethylene oxide in rodents after inhalant exposure.

GLP compliance:

not specified

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Supplier: Fluka Chemical (Ronkonkoma, NY, USA)
- Physical appearance: gas
- Purity: > 99.9%

ISOTOPE INFORMATION
- Supplier: Cambridge Isotopes (Cambridge, MA, USA)
- Specificity: [1,2-C13]ethylene oxide

Radiolabelling:

yes

Species:

other: rats and mice

Strain:

other: Fischer 344 and B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (Raleigh, NC, USA)
- Age at study initiation: Rats: 10 -12 weeks; mice: 7 - 9 weeks
- Weight at study initiation: Rats: 245 +/- 10 g (male), 186 +/- 7 g (female); mice: 25.8 +/- 1.4 g (male), 20.8 +/- 0.9 g (female)
- Housing: individually
- Diet: pelleted rodent diet, ad libitum
- Water: deionized water, ad libitum
- Acclimation period: at least 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 2
- Humidity (%): 55
- Photoperiod (hrs dark / hrs light): 12

Route of administration:

inhalation: gas

Vehicle:

other: filtered air

Details on exposure:

TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: whole-body exposure. 31-l Leach chamber (4x4x10 inch)
- Temperature, humidity, pressure in air chamber: 22°C, 55%
- Air flow rate: 10 liters/minute

TEST ATMOSPHERE
- Brief description of analytical method used: Chamber concentrations were monitored continuously at both the inlet and outlet with an infrared spectrometer at a wavelength of 11.8 µm

OTHER SPECIFICS.
Three exposures were conducted at each concentration for each sex and species

Duration and frequency of treatment / exposure:

4 h

Dose / conc.:

100 ppm

Dose / conc.:

330 ppm

No. of animals per sex per dose / concentration:

5

Control animals:

not specified

Details on study design:

- Dose selection rationale: Concentrations were chosen to bracket the threshold exposure concentrations (approximately 100 ppm, 4 hrs) at which GSH depletion has been reported to occur in both rats and mice following acute EO exposure.
- Headspace determination in mice and rat blood

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: muscle, brain (rat only), blood, testis, liver, kidney, lung
- Time and frequency of sampling: Rats were killed 2 and 20 minutes post-exposure
- Other: Tissue weights were determined gravimetrically
- Enzyme kinetics: glutathione S-transferase, epoxide hydrolase

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: tissues
- Time and frequency of sampling: 2 and 20 minutes post-exposure
- From how many animals: samples
- Method type for identification: NMR

Statistics:

- ANOVA using student's t test: intra- and interspecies differences between in vitro kinetc constants. Significant if p ≤ 0.05; differences between in vivo rate constats for different sexes, species, and exposure levels
- Welch's approximate t test: compare mean steady-state tissue concentrations. Significant if p ≤ 0.05

Preliminary studies:

no preliminary studie performed

Type:

metabolism

Results:

Assumed to be transformed entirely by cytosolic GST (cGST) in liver and kidney

Type:

excretion

Results:

Mice eliminated ethylene oxide much more efficiently than rats

Details on absorption:

not determined

Details on distribution in tissues:

Results demonstrate that EO is rapidly distributed. Testicular EO concentrations were 20-50% lower than blood levels.

Details on excretion:

The clearance of EO from blood of mice was more rapid than from the blood of rats.
t(1/2) in rats: 13.8 +/- 3.0 and 10.8 +/- 2.4 minutes for males and females, respectively
t(1/2) in mice: 3.12 +/- 0.2 and 2.4 +/- 0.2 minutes for males and females, respectively
Steady-state had been achieved. Steady-state tissue levels were below 100 µM (mice). No saturation of metabolism was observed in rats.

Toxicokinetic parameters:

other: V(max) rat

Remarks:

20.3 - 52.7 nmol/mg protein/min

Toxicokinetic parameters:

other: V(max) mouse

Remarks:

17.3 - 287 nmol/mg protein/min

Toxicokinetic parameters:

other: KM rat

Remarks:

8.1 - 13.0 mM

Toxicokinetic parameters:

other: KM mouse

Remarks:

7.1 - 11.0 mM

Metabolites identified:

yes

Details on metabolites:

- S-(2-hydroxyethyl)glutathione (2-HEG)
- ethylene glycol (EG)
- 2-chloroethanol (2-CE)

Bioaccessibility (or Bioavailability) testing results:

not determined

The time-weighted average chamber concentrations were 99 +/- 2 and 327 +/- 2 ppm.

Gender differences were only observed in mice. Female mice had a significantly higher concentration at the end of exposure (C(0)) than male mice. Female rats had a significantly lower cGST Vmax in liver than males. However, the Vmax/KM for rat liver cGSTs were similar.

Three metabolites have been detected in mouse and rat liver cytosol following incubation with glutathione and [13C]ethylene oxide, as stated above.

Conclusions:

The results presented here suggested a marked interspecies differences in the efficiency of EO elimination in rodents, while sex differneces are minimal. There appears to be a trend toward decreased ability to clear EO with increasing animal size.

Executive summary:

Ethylene oxide (EO) is a direct-acting mutagen and animal carcinogen used as an industrial intermediate and sterilant with a high potential for human exposure. Kinetics of EO metabolism in rodents can be used to develop human exposure. Kinetics of EO metabolism in rodents can be used to develop human EO dosimetry models. This study examined the kinetics of EO metabolism in vivo and in vitro in male and feamles F-344 rats and B6C3F1 mice. In vivo studies measured blood and tissue EO levels during and 2 -20 min following whole-body inhalation exposure (4 h, 100 or 330 ppm EO). At 100 ppm, the half-life of elimination (t(1/2)) in rats was 13.8 +/-0.3 (mean +/- SD) and 10.8 +/- 2.4 min for males and females, respectively, compared to a t(1/2) in mice of 3.12 +/- 0.2 and 2.4 +/- 0.2 min in males and females, respectively. On exposure to 330 ppm, the t(1/2) in miceincreased approx twofold, while no change in t(1/2) was ibserved in rats. In vitro kinetic parameters (V max and KM) of EO metabolism were determined using tissue cytosol and microsomes. EO metabolism in vitro occurred primalrily via cytosolic glutathione S-transferase-mediated EO-GSH conjugation (c-GST-EO), with highet activity in the liver. Liver cGST-EO activity (Vmax) was 258 +/- 86.9 and 287 +/- 49.0 nmol/mg protein/min (mean +/- SD) in male and female mice, respectively, compared to 52.7 +/- 10.8 and 29.3 +/- 4.9 in male and female rats, respectively. In rats, but not in mice, there was a statistically significant (p<0.05) gender difference in the Vmax for liver cGST. The KM for liver cGST-EO was approx 10 mM in both species. The higher Vmax values observed in mice are consistent with the more rapid elimination of EO observed for this species in vivo compared to rats.

Description of key information

McKelvey and Zemaitis (1986): non-guideline, non-GLP study in male Fischer 344 rats and male Swiss-Webster mice. Animals were exposed for 4 -hr to 100 (mice/rat), 400 or 900 ppm for mice and 100, 600 or 1200 ppm for rats vapor. Results indicate a marked species regarding effects on blood GSH levels. No mortality was reported neither in mice nor in rats. 

Brown et al. (1996): non-guideline study using Fischer 344 rats and B6C3F1 mice. GLP not specified. Animals were exposed for 4 -hr to 100 and 330 ppm vapor. No mortality was observed neither in mice nor in rats. Results indicated that ethylene oxide is transformed entirely by cytosolic GST (cGST) in liver and kidney. Mice eliminated the substance more efficiently than rats.

A number of other kinetic studies including toxicokinetic models for exogeneous and endogeneous EO metabolism is reported in IUCLID chapter 7.12.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

100

Absorption rate - dermal (%):

1.3

Absorption rate - inhalation (%):

100

Additional information

Absorption:

Oral:

Signs of toxicity were observed and thus, absorption through the oral route is confirmed. Smyth et al. (1941) confirmed the oral route being relevant with an acute toxicity study in Wistar rats leading to adverse effects, such as sluggish depressed functioning and death. Applying Lipinski’s rule of five, the potential of EO being orally bioavailable was strongly supported. No absorption rate has been mentioned in any study and thus, an absorption rate of 100% has to be assumed.

Inhalation:

Inhalation is suggested to be one of the most relevant routes of exposure due to its physical chemical properties. EO is a highly volatile substance and favorable for absorption directly across the respiratory tract epithelium by passive diffusion. Based on its rather small size it reaches deep into the respiratory tract. Various authors reported adverse effects, such as tremor, effects to the central nervous system and cancer. A specific absorption rate has not been mentioned and thus, is assumed to be 100%.  

Dermal:

Dermal absorption is suggested to be the least crucial route, because the substance is highly volatile. In general, a molecular mass of less than 100 g/mol favors dermal uptake and the chemical structure does not reveal an ionic potential slowing the uptake through the skin. On the other hand, the low log P < -1 suggests that crossing the stratum corneum is unlikely due to insufficient lipophilicity. Accordingly, an absorption rate was reported by Bader et al. (2012) being only 1.3%. However, in an incident where three operators were exposed during repairs of a leakage while wearing independent breathing apparatus providing a high level of respiratory protection, haemoglobin adducts have been found in the blood one to two orders of magnitude over the Dutch OEL of 0.5 ppm. Thus, EO exposure was almost certainly caused by dermal uptake of EO vapor. EO was shown to be able to penetrate the skin as vapour: the percutaneous absorption of EO from fabric was 46% of an experimental dose if the source material (fabric) was applied in an occluded setting onto the skin (e.g. inside a glove), whereas only 1.3% of the dose was absorbed when the fabric/skin surface was open to surrounding air (Boogaard, 2014).

The dermal absorption has been shown in one publication by Bader et al. (2012) reporting one worker who was accidentally exposed to EO experiencing a sunburn-like itchy feeling on the skin. In addition, ethylene oxide exposure (gaseous and liquid state) to the skin showed toxic dermatitis with potent blistering (Thiess 1963).

 

Distribution:

Based on the results obtained from experimental animals, EO was found in various tissues such as muscles, brain, blood, and testis after inhalant exposure (Brown, 1996). It was demonstrated that EO is rapidly distributed throughout sites. The substance crosses membranes in order to reach the target organs, such as liver and kidney. On the other hand, effects on the central nervous system lead to the conclusion that the substance is not only transported via passive mechanisms, but also actively by transport proteins in order to cross the blood-brain-barrier. Ethylene oxide has also been found in slowly equilibrating tissues, e.g. muscle.  

 

Metabolism:

Brown et al. (1996) conducted a toxicokinetic study in Fischer 344 rats addressing the question of metabolism. Results showed that EO was metabolized via phase I as well as phase II enzymes, namely epoxide hydrolase (EH) and glutathione S-transferase (GST). EO metabolites were the glutathione conjugate S-(2-hydroxyethyl)glutathione as well as the hydrolysis products ethylene glycol and 2-chloroethanol. The main metabolizing organ was found to be the liver followed by kidney and testes. Brain and lung contained only slight activity relative to the other tissues. Hence, it was concluded that EO was assumed to be catalyzed entirely by cytosolic GST (cGST) in liver and kidney. Contributions from chemical hydrolysis and nonenzymatic GSH conjugation were reported to be slight. Further, several adducts are formed which have been quantitively analyzed in diverse studies. N-(2-hydroxyethyl)valine (HOEtVal), a haemoglobin adduct, and the DNA adductN7-(2-hydroxyethyl)guanine (N7-HEG) are two clear biomarkers of exposure.

 

Excretion:

Brown et al. (1996) investigated the elimination of EO from blood in rats and in mice. It was confirmed that EO follows a first-order kinetic with a half-life in rats and mice (mean value) of 12.3 minutes and 2.8 minutes, respectively. The half-life in humans was estimated to be approximately 40 minutes (Filser, 1992 and Brown, 1996). According to the low half-life and the low log P value of -3 it is assumed that EO does accumulate only to a negligible degree in mammal tissue. Marked interspecies differences in the clearance of EO in rodents were reported by Brown et al. (1996) while sex differences were only minimal. It was suggested that there is a trend toward decreased ability to eliminate the substance with increased animal size.