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Basic toxicokinetics

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basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference Type:
The effects of Ethylene Oxide exposure on tissue glutathione levels in rats and mice.
McKelvey, J.A. and Zemaitis, M.A.
Bibliographic source:
Drug Chemical Toxl 9(1), 51-66

Materials and methods

Objective of study:
Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethylene oxide
EC Number:
EC Name:
Ethylene oxide
Cas Number:
Molecular formula:
not specified

Test animals

other: rats and mice
other: Fischer-344 and Swiss-Webster
Details on test animals or test system and environmental conditions:
- Weight at study initiation: Male rats (180 - 210 g) and male mice (28 - 32 g)
- Diet: ad libitum
- Water: ad libitum

Administration / exposure

Route of administration:
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
Doses / concentrationsopen allclose all
Dose / conc.:
100 ppm
Dose / conc.:
400 ppm
Dose / conc.:
900 ppm
Dose / conc.:
600 ppm
Dose / conc.:
1 200 ppm
No. of animals per sex per dose / concentration:
Groups of 4 rats or 4 - 20 mice
Control animals:
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.
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.

Results and discussion

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

Toxicokinetic / pharmacokinetic studies

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

Metabolite characterisation studies

Metabolites identified:
not measured
Details on metabolites:
not determined

Bioaccessibility (or Bioavailability)

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%.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.