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Repeated dose toxicity: oral

In 1956, Hollingworth et al briefly reported on a subacute gavage study in rats and this information is assigned a validity score of 4. A twenty-one-day exposure resulted in irritation of the stomach, effects on body weight and liver at 100 mg/kg bw. Local adverse effects on the stomach after gavage dosing are consistent with the corrosivity. Adverse findings were not reported for the lower dosages (3 and 30 mg/kg bw.).

Repeated dose toxicity: inhalation

Snellings et al. (1984) conducted a subchronic study in 30 B6C3F1 mice per sex per dose which was equivalent or similar to OECD 413 with deviations concerning the exposure time of 10 and 11 weeks. GLP compliance was not specified. Male and female animals were given concentrations of 10, 50, 100 and 250 ppm for 6 hours per day and 5 days per week for a study duration of 10 and 11 weeks for males and females, respectively. No mortality was observed. A dose-related trend of response in the 250, 100, and 50 ppm exposure groups was noted in the evaluation of locomotor functions; however, because of the small sample size, determination of what concentrations were effect or no-effect levels is difficult. The NOAEC was found to be 10 ppm (equivalent to 18 mg/m3) for male and female mice.

 

Bushy Run Research Centre (1982) conducted a subchronic GLP-conform study in 35 Fischer 344 rats per sex per dose and 15 CD1 and CF1 mice per sex per dose which was equivalent or similar to OECD 413 with deviations concerning the exposure time of 7 and 8 weeks. The animals were given vapour concentrations of 50, 100, 150, and 450 ppm (equivalent to 91, 183, 275, and 824 mg/m3) for 6 hours per day and 5 days per week over a study period of 7 - 8 weeks. Within 2 to 3 weeks, high numbers of mortalities and other significant treatment-related effects for both rats and mice occurred at the 450 ppm exposure level. Before death occurred in rats and mice of this exposure group, observations of tremors, convulsions, and paresis of the hindquarters were observed in several animals. Although there was no clear pathogenesis, the most probable cause of death for the rats was vascular damage or nasal cavity obstruction. Histologic changes noted for the rats of the 450 ppm concentration group, which were sacrificed after 2 or 3 weeks of exposure, were various lesions in the nasal cavity mucosa, lymphoid tissue atrophy, and testicular degeneration. Transient depression in the rats of gain in body weight was observed for male rats and male mice exposed to concentrations as low as 50 ppm of ethylene oxide. Only a few significant findings in organ weight determinations and clinical pathology values were noted in the rats of the 150 ppm exposure level; however, none of these were supported by histopathologic alterations. A NOAEC was not established.

 

Lynch et al. (1984) performed a chronic non-guideline, non-GLP study in 60 adult male monkeys. The animals were exposed to vapour concentrations of 50 and 100 ppm for 7 hours per day and 5 days per week over a study period of 2 years. The 100 ppm group had a statistically significant reduced mean body weight compared to the control group beginning at week 19 and continuing through week 104. Five monkeys died during the 2-year exposures, one each in the ethylene oxide 50 and 100 ppm groups. These deaths did not appear to be related to oxide exposure. However, a NOAEC was not determined.

Repeated dose toxicity: dermal

No study available for systemic effects upon repeated dermal exposure. The substance is corrosive to skin and a skin sensitizer.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
chronic toxicity: inhalation
Remarks:
combined repeated dose and carcinogenicity
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
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
- Supplier: Union Carbide Corporation
- Physical appearance: liquid
- Purity: > 99.9%
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
Age of animals: 6 weeks
General health status was determined during a 2-week quarantine period. Rats accepted were those for which no abnormal clinical signs were observed and whose body weights were within plus or minus two standard deviations of the mean weight of all rats of the same sex.
All animals were housed and exposed, five per cage, in wire-mesh stainless steel cages. Light/dark cycle: 12 h. Animals were exposed during the light period at approx. the same time each day. The temperature and humidity controlling devices of the room were set to maintain the environment between 20 and 24°C and 40 - 60% rel. humidity. Feed and water were removed during the exposure period but were available ad libitum at all other times.
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
Exposure and analytical systems. A stainles-ssteel cylinder containing liquid ethylene oxide was heated to approximately 35°C. The vapor generated was metered into 4400-liter stainless-steel and glass inhalation chambers. Chamber temperature and relative humidity were controlled, and the values for these parameters were recorded during each exposure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber atmospheric concentrations in both the test and control chambers were monitored by a gas chromatograph equipped with a fame ionization detector. The column was packed with Tergitol-TMN surfactant (30%) and sodium methylate (3%) on Chromosorb W-NAW support material (Klein et al., 1983). Approximately eight analyses were obtained from each chamber within each 6-hr exposure period.
Duration of treatment / exposure:
6 - 24 months
Frequency of treatment:
6 h/d, 5 d/w
Dose / conc.:
10 ppm (nominal)
Remarks:
18 mg/m³
Dose / conc.:
33 ppm (nominal)
Remarks:
60 mg/m³
Dose / conc.:
100 ppm (nominal)
Remarks:
183 mg/m³
No. of animals per sex per dose:
120
Control animals:
yes, concurrent no treatment
Details on study design:
Two air control groups were exposed under similar conditions.
Whole body exposures were conducted in a dynamic exposure system where the vapor concentration levels were determined by gas chromatography.
Observations and examinations performed and frequency:
After initiation of exposure, animals were routinely examined for signs of toxic effects including body weight determination and palpation for abnormal tissue masses. After 6 and 12 months of exposure, 10 rats per sex per dose were necropsied. After 18 months, 20 rats per sex per dose were necropsied, and after 24 months (females) or 25 months (males), all remaining rats were necropsied.
Sacrifice and pathology:
A complete necrospy was performed on each animal killed at a scheduled necropsy interval ans on all animals which were found dead or moribund. At the 6-month and final intervals, approx. 50 tissues from each of the rats in the 100-ppm and two air control groups were histopathologically examined (details not mentioned). The same tissues were examined from all rats that died or were killed in a moribund condition from any group. At the 12- and 18-month intervals, app. 15 major organs and tissues were microscopically examined from the rats in the 100-ppm and both control groups. Only tissues with gross lesions were examined from the rats in the 33- and 10-ppm groups at the 6-12-, and 18-month intervals. At the final interval, approx. 20 major organs and tissues were examined from the rats in the 33- and 10-ppm groups. Tissues saved were fixed in 10% neutral buffered Formalin. Tissues submitted for histopatholigic evaluation were stained with hematoxylin and eosin.
Statistics:
Tumor incidence data for each exposure group were compared statistically to the combined data of the two air control groups. For all statistical comparisons, 2-tailed probabilities are reported. For all life table statistical comparisons, the critical ratios 2 were calculated using standard errors. The probabilities associated with the critical ratios and with the results of the Fisher's exact comparisons for tumor incidences were adjusted by the Bonferroni correction to account for the multiplicity of comparisons. Additional statistical tests for positive trends were also used. Time-adjusted trend test analyses were performed to compensate for bias associated with differential mortality in the various treatment groups. The adjusted analyses are sensitive not only to differences in relative tumor frequencies among groups but also to the time of observation of the tumors. Thus, the trend tests indicate not only whether the treatment results in more tumors but also if tumors develop earlier. An additional statistical analysis was also performed. In this test, early deaths are eliminated from the procedure as described by Gart et al.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
Exposure concentrations
The actual daily ethylene oxide concentration within the chambers were very close to the target concentration of 100, 33 and 10 ppm. The overall 2-year means (with the coefficient of variation) of the daily measured chamber concentration were 101 (5%), 33 (3%) and 10 (5%) ppm for the high, intermediate, and low exposure concentration.
A viral sialodacryoadenitis (SDA) occurred during the 15th exposure month and exposures to EtO were temporarily stopped for 2 weeks. During this time, body weight gain and almost all clinical signs returned to preinfection status.

Body weights
A statistically significant depression in body weight gain was noted in the 100-ppm group for both male and female rats beginning about the end of the first exposure month. Following 10 weeks of exposures to 33 ppm of EtO, an effect on the body weight of only the female rats was observed. These treatment-related effects were generally observed throughout most of the remainder of the study. Furthermore, for both sexes, the body weight curves of the two control groups and the 10-ppm groups were similar throughout the study.

Mortality
Very low mortality had been observed prior and after SDA outbreak. During viral infection one or more animals of each group died. However, after 2 weeks of no EtO exposure, the rate of mortality decreased to the level prior to the infection.
Since the 20th exposure month, mortality increased and was significantly higher for both sexes in the 100- ppm group compared to control groups. From the 21st month on, the values for both sexes of the 33-ppm group were numerically greater than those of both control groups.

Based on these results the No-observed effect concentration was determined to be 10 ppm. For details on carcinogenic effects, please refer to IUCLID section 7.7.
Dose descriptor:
NOAEC
Effect level:
10 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: NOAEC for clinical effects; carcinogenicity not covered by NOAEC.
Critical effects observed:
no
Endpoint:
sub-chronic toxicity: inhalation
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
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
exposure period of 10 and 11 weeks
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
- Physical appearance: liquid
- Purity: > 99.9%
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratory
- Fasting period before study: No
- Housing: Animals were housed two mice per cage
- Diet: ad libitum except during inhalation exposure period
- Water: ad libitum except during inhalation exposure period
- Acclimation period: 4 weeks
Route of administration:
inhalation: vapour
Type of inhalation exposure:
not specified
Vehicle:
other: no vehicle
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Stainless steel and glass inhalation chamber
- Temperature and humidity: 22 to 26 °C, 40 to 60 %
- Air flow rate: 1800 L/min

TEST ATMOSPHERE
- Brief description of analytical method used:
Ethylene oxide concentrations in both the test and control chambers were monitered by a gas chromatograph equipped with a flame ionization detector. The column was packed with Tergitol-TMN surfactant (30%) and sodium methylate (3%) on Chromosorb W-NAW support material.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Approximately eight analyses were obtained from each chamber within each 6-hr exposure period.
Duration of treatment / exposure:
10 weeks for males, 11 weeks for females
Frequency of treatment:
6 h/d, 5 d/w
Dose / conc.:
10 ppm (nominal)
Remarks:
18 mg/m³
Dose / conc.:
50 ppm (nominal)
Remarks:
90 mg/m³
Dose / conc.:
100 ppm (nominal)
Remarks:
180 mg/m³
Dose / conc.:
250 ppm (nominal)
Remarks:
450 mg/m³
No. of animals per sex per dose:
30
Control animals:
yes, concurrent no treatment
Details on study design:
Four groups of B6C3F1 mice were exposed to ethylene oxide. A fifth group, handled similarly as the ethylene oxide-exposed groups, was exposed to ethylene oxide-free air. After an exposure regimen of 6 hours per day, five days per week, for ten weeks (males), or eleven veeks (females), urine and blood samples were submitted for clinical pathologic evaluation and selected tissues were weighed and histopathologically examined.
Observations and examinations performed and frequency:
All mice were weighed weekly and observed throughout the exposure period for any signs indicative of a toxic effect.
At termination, 10 mice per sex per group were used for urinalysis, hematology, serum clinical chemistry, organ weight and necropsy evaluations. Semiquantitative analysis of the urine included determinations for bilirubin, glucose, ketones, nitrite, occult blood, pH, protein, and urobilinogen. To complete the blood analyses and necropsy work each day within a short period of time, an equal number of mice of the same sex from each group were randomly selected to be examined daily over a 3-day period. All animals were exposed for a minimum of two consecutive daily exposure periods before blood and urine were collected and the animals were killed.

Approximately 18 hr following the termination of the last exposure, animals were lightly anesthetized with methoxyfluorane and blood was obtained from the retroorbital sinus. The blood sample collected for hematologic evaluation contained ethylenediaminetetraacetic acid as an anticoagulant.

Hematologic analysis included determination of red blood cell count (RBC), packed cell volume (PCV), hemoglobin (Hgb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), white blood cell count (WBC), and leukocyte differential count.

Serum clinical chemistry measuremnents were made for glucose, blood urea nitrogen, creatinine, protein, albumin, alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase.
Sacrifice and pathology:
Following blood collection, mice were exsanguinated by transection of the brachial blood vessels. After a thorough gross tissue examination at necropsy, selected organs were weighed and fixed in 10% neutral buffered Formalin. The following tissues were then sectioned and stained with hematoxylin and eosin for histopathologic evaluation: sciatic nerve, gastrocnemius muscle, brain, and spinal cord from both sexes; and testes from males; and liver, sternal marrow, and spleen from females.
Other examinations:
A neuromuscular screening test was performed to determine the feasibility of conduction larger scale neurologic examinations in subsequent studies. The screening test used was a modification of a comprehensive observational assessment of the behavioral and physiologic state of the mouse which was first described by Irvin (1966). Observations included locomotor activity, patterns of respiration, corneal response, gait, tail and toe pinch reflex, and righting reflex. Independently at each examination period, five mice were randomly selected for testing. The test was performed in a blind fashion immediately following an exposure.
Statistics:
The fiducial limit of 0.05 (two tailed) was selected as the critical level of significance. For continuous variable, contingency, and nonparametric data, the same statistical procedures as those presented by Snellings et al. (1982) were followed.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
The overall means (with cofficient of variation) of the daily measured chamber concentrations were 236 (4%), 104 (4%), 48 (4%), and 10 (6%) ppm, respectively, and were close to the target concentrations.
There were no treatment-related effects on survival or body weight for either male or female mice; nor were there any consistent treatment-related clinical signs of toxicity noted.
Statistically significant differences to control animals were observed in the neuromuscular screening test. In the 250-, 100-, and 50-ppm exposure groups, there were statistically significant differences noted for abnormal posture (hunched), during evaluation of gait, and reduced locomotor activity. For abnormal reflexes of righting, toe pinch, and tail pinch, statistical differences were observed for the most part only in the 250 ppm- exposure group.
Hematology revealed a depression in red blood cell count, packed cell volume, and hemoglobin concentration for both sexes in the 250- ppm exposure group. At this exposure concentration, there was no apparent effect on either the total or differential white blood cell count. There were no treatment-releated hematologic effects at the lower exposure concentration. The serum clinical chemistry values for both sexes did not indicate any treatment- related effect. Likewise, semiquantitative analysis of the urine did not reveal any effects related to ethylene oxide exposure.
No biologically significant, treatment-related effects were noted for the kidneys, adrenals, and thymus. There were statistically significant differences between the means of treated and control animals for the following organs in the 250-ppm exposure group: the liver weights (relative) of the female mice were elevated; the testicular weights (absolute) were depressed; and the spleen weights (absolute and relative) of both sexes were depressed. For the rest of the exposure groups, the mean spleen weight (absolute and relative) of the 100-ppm- exposed females was the only other value that was significantly different (depressed) from the control.
Histologic sections of liver, testes, spleen, and brain were normal. Also, there were no abnormalities noted on the light microscopic examination of the spinal cord, sciatic nerve, gastrocnemius muscle, and sternal marrow from mice in the 250-ppm exposure group.
Results of a neuromuscular function test indicated that certain reflex responses and locomotor activities were affected in the ethylene oxide-exposed animals. A dose-related trend of response in the 250, 100, and 50 ppm exposure groups was noted.
Based on these results the No-observed effect concentration was determined to be 10 ppm.
Dose descriptor:
NOAEC
Effect level:
10 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: alterations in locomotor activity and reflex responses
Critical effects observed:
yes
Lowest effective dose / conc.:
50 ppm (nominal)
System:
central nervous system
Organ:
brain
Treatment related:
yes
Dose response relationship:
yes

Table 1

Percentageaof Responders in a Neuromuscular Screening Test in B6C3F1Mice exposed to Ethylene Oxide for 10 to 11 weeks

Intervalb

Sex examined

Target exposure concentration (ppm)

250

100

50

10

0

 

 

Reduced or no toe pinch reflex

 

 

 

Intermediate

F

100**

40

40

40

0

Terminal

F

--c

--

0

--

0

Terminal

M

60

20

20

0

0

 

 

Reduced or no tail pinch reflex

 

 

 

Intermediate

F

60

60

20

40

20

Terminal

F

60

40

20

--

0

Terminal

M

100**

40

60

0

0

 

 

Abnormal righting reflex

 

 

 

Intermediate

F

100*

100*

20

40

20

Terminal

F

80*

0

0

--

0

Terminal

M

80*

60

20

0

0

 

 

Hunched posture during gait

 

 

 

Intermediate

F

100*

100*

40

20

20

Terminal

F

100**

80*

80*

--

0

Terminal

M

100**

100**

40

20

0

 

 

Reduced locomotor activity

 

 

 

Intermediate

F

100

60

40

20

60

Terminal

F

100**

100**

60

--

0

Terminal

M

100**

100**

80*

40

0

 

a    N = 5.

b    Intermediate interval occurred during the sixth exposure week and terminal interval was just prior to sacrifice.

c    Not determined

*    p < 0.05 in comparison to control group

**        p < 0.01 in comparison to control group

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
exposure duration 7 to 8 weeks
GLP compliance:
yes (incl. QA statement)
Remarks:
testing lab.
Limit test:
no
Specific details on test material used for the study:
- Supplier: Union Carbide Corporation
- Physical appearance: liquid
- Purity: > 99.9%
Species:
other: rats and mice
Strain:
other: Fischer 344 (rats), CD1 and CF1 (mice)
Sex:
male/female
Details on test animals or test system and environmental conditions:
Male and female rats were obtained from Microbiological Associates, Inc. (Walkersville, MID) and male and female mice were obtained from Charles River Breeding Laboratories, Wilmington, MA and Saint Constance, Ontario, Canada, respectively.
The Fischer 344 rat strain was selected because of its intended use in a two-year ethylene oxide vapor study. The reasons for selection of this strain included (i) fact that this strain has been accepted for the carcinogen bioassay program of the National Toxicology Program, (ii) it had been widely accepted in chronic inhalation studies partially due to its small size which lessen the heat load in the inhalation chamber, and (iii) because of its good longevity record in chronic studies. Two strains of mice were selected to determine if there were any appreciable differences in the toxic responses between strains of mice. At the laboratory, both strains have been used in previous toxicology studies.
All animals were approximately 4 weeks old upon arrival at the laboratory and appeared in good health. Quality control examinations which included body weight determinations, fecal examination for intestinal parasites by zinc sulfate flotation, evaluation of the nasal larynx and lung for aerobic bacteriologic flora, and micropathological examination of selected tissues were performed on a portion of the rats upon receipt. The eyes of all rats were grossly examined (visual examination) for lesions prior to the start of the exposures. The results of the quality control and microscopic examinations were within normal limits for commercially available, specific pathogen-free rats; therefore, the animals were determined to be of suitable quality for the inhalation study. Only limited quality control evaluations were performed on the mice since the main objective of this study was to evaluate the effects of ethylene oxide vapor exposure on rats.
All animals were identified with individual numbers by a sequential toe-clipping procedure. Listed on each color-coded cage card (identifying exposure concentration) was the identification numbers of the animals within the cage.
The animals were observed for two 2eeks prior to assignment into exposure groups. During this period, all animals were weighed three to four times and assigned to exposure levels using a card-based randomization system. An animal was not used for group assignment if it were not gaining weight
normally, if abnormal clinical observations were noted, or if the body weight at the time of group assignment were above or below to standard deviations from the mean of all animals of that sex.
The rats were housed five per cage and mice were housed three per cage in suspended, stainless steel, wire mesh cages, 35 cm x 37 cm x 18 cm high for rats and 35 cm x 17 cm x 18 cm high for mice (separated by test group and sex). The animals were housed in the same cages during exposure and non-exposure periods. When the animals were in the room, the temperature and humidity controlling devices of the non-recirculated air supply to the room were set to maintain the environment between 66 and 77 °F and 30 to 70% relative humidity. The fluorescent lighting was set on a 12-hour photoperiod. Water was available, ad libitum, to the rats and mice throughout the exposure and non-exposure periods by an automatic watering system. Purine diet was available ad libitum throughout the non-exposure period only.
A stainless steel shelf pan was placed between each level of cages to prevent urinary and fecal contamination of the animals on lower tiers. These pans were in place during the exposures. After each exposure, clean pans with absorbent paperboard were placed on the carriers. Before exposure, the absorbent paperboard was removed.
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
The animals were exposed to ethylene oxide vapor or room air in 3800-liter stainless steel-lined inhalation chambers. Each chamber had glass windows for observation of animals. Chamber temperature and relative humidity were recorded three times a day on average. The airflow through the chambers was maintained at approximately 1000 liters per minute.
Liquid aliquots (approximately 5 pounds) from the storage drum were transferred to a stainless steel cylinder. This cylinder which was attached to the generation system was maintained at approximately 30°C by mneans of a constant temperature recirculating bath. The ethylene oxide vapor pressure generated at that temperature was utilized to conduct the gas through stainless steel tubing and a pressure-reducing regulator to maintain approximately three psi internal pressure. Manifolds of stainless steel tubing directed the vapor through control values and flowmeters to the chamber air-inlet duct. Ethylene oxide vapor was diluted at the inlet duct with exposure room air and drawn into the inhalation exposure chamber.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All chambers were monitored for ethylene oxide concentration by means of a Varian 2700 gas chromatograpgh (GC). The chamber atmosphere samples were manually injected into the GC and an automatic recording system connected to the GC transferred the data to a printer and onto magnetic trap. A known primary standard concentration of ethylene oxide was sampled several times before the start of daily chamber analysis to check the monitoring system. Each day, approx. four samples were analysed from each ethylene oxide-exposure chamber; and between one and three samples of the air within the exposure room and a similar number of samples from the control chamber were analysed.
Duration of treatment / exposure:
7 - 8 weeks
Frequency of treatment:
6 h/d, 5 d/w
Dose / conc.:
50 ppm (nominal)
Remarks:
90 mg/m³
Dose / conc.:
100 ppm (nominal)
Remarks:
180 mg/m³
Dose / conc.:
150 ppm (nominal)
Remarks:
270 mg/m³
Dose / conc.:
450 ppm (nominal)
Remarks:
810 mg/m³
No. of animals per sex per dose:
Each of the groups contained 35 male and 35 female Fischer 344 rats und 15 male and 15 female CD-1 und the same number of CF-1 mice, with the exception of the control group which contained an additional 10 male and 10 female rats. Included in this number of rats were "extra" rats for each group. The ethylene oxide exposure groups contained 7/sex and the control group contained 17/sex. These rats were to be used if an unscheduled necropsy interval was necessary. If these additional rats were not used, they were sacrificed and removed from the study at the termination of the exposures for that group. Necropsy was not performed for these rats.
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Target concentrations of 450, 150, 100, and 50 ppm of ethylene oxide were selected for the exposure concentrations based on results of previous rat and mouse studies as reported by Hollingsworth, et al. (1956) and Jacobson, et al. (1956). All animals were exposed for six hours per day, five days per week; the male rats received 36 exposures, female rats received 37 exposures, and both sexes and strains of mice received 33 exposures. The rats had only four exposures during the first week. Control (air-exposed) animals were handled in an identical manner as the ethylene oxide-treated animals. Animal cages were rotated within each chamber on a weekly basis to compensate for any possible, but undetected, variation in chamber exposure conditions (i.e., concentration, temperature, and/or relative humidity). Because of high mortality, exposures for the 450 ppm group were terminated, following 14 exposures for rats and 11 exposures for mice.
Observations and examinations performed and frequency:
During the six-hour exposure, a portion of the animals were observed several times through the chamber windows. However, most changes in general health status were better observed for all animals immediately before and after the exposure. Any abnormalities in appearance were recorded at these times.
A neuro-muscular function examination was performed on the male and female rats of the 150 ppm and control groups following 18, 19, 21, 23, 26, 28, 31, 33, and 37 completed exposures. [Note: This is an addition to the protocol]. The same five rats per sex per group were evaluated at each examination period, with the exception of the final examination where 12 female rats per group and 7 males from the 150 ppm group and 8 control group males were examined. The examination that occurred following 33 completed exposures only consisted of examining the righting reflex and climbing ability of the rate.
All animals were weighed the morning before the first exposure. This weight, termed the pre-exposure reference weight, was subtracted from each subsequent weight determination to obtain the change in body weight. Body weights were measured weekly throughout the study, rats an Friday and mice an Tuesday. In addition, all animals were weighed at the time of sacrifice.
Urine (rats only) was collected for approximately two hours from both male and female rats prior to the interim and final sacrifice periods. The urine was semi-quantitatively analysed by a dip stick method. The items evaluated were as follows: bilirubin, glucose, ketones, nitrite, occult blood, pH, protein, and urobilinogen. Specific gravity was determined by using a refractometer. Urine volumes were not measured.
Hematologic and serum clinical chemistry evaluations (rats only) were performed on randomly-selected male and female rats at three and eight weeks of exposure, and on two days preceding each sacrifice period. At an unscheduled sacrifice during the second exposure week, only blood from female rats of the 450 ppm and control groups were submitted for evaluation.
All rats had free access to food and water prior to evaluation but were deprived of both food and water during the bleeding period. The rats were lightly anesthetized with methoxyflurane and blood was collected from the interior vena cava. After blood collection, the animals were sacrificed by cervical dislocation.
Other than the samples used for clotting time determinations, all samples for hematology were collected in heparinized tubes. Hematologic parameters included white blood cell count , white blood cell differential count, red blood cell count, packed red cell volume, hemoglobin concentration, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration and clotting time. Clotting was determined by drawing a needle through a fresh drop of blood until a strand was noted. Clotting time was also determined for the male CD-1 and CF-1 and female CF-1 mice following 10 exposures (450 ppm and control only) and 33 exposures (150 ppm and control only). The CD-1 female mice of the 450 ppm exposure group were all dead by the tenth exposure, consequently, clotting times for the CD-1 female mice of the control group were not determined.
The biochemical analysis included cholesterol, serum urea nitrogen, creatinine, lactic dehydrogenase, alkaline phosphatase, albumin, creatine phosphokinase, alpha-hydroxybutyric dehydrogenase, serum glutamic oxaloacetie transaminase, serum glutamic pyruvic transaminase, calcium, glucose, cholinesterase, total bilirubin, total protein, and glutamyl transpeptidase.
Sacrifice and pathology:
At each sacrifice period, the rats and mice were killed by severing the cervical spinal cord and exsanguination via the jugular und carotid vessels. For all rats, the sciatic nerve was dissected free und fixed in 3% phosphate buffered glutaraldehyde for possible future examination. The femur was removed and a fresh bone marrow impression smear was prepared and stained with Wright-Giemsa stain for each rat. All viscera from these animals were removed and representative samples were fixed in 10% neutral buffered formalin. The lungs were weighed and then inflated with 10% NBF. The eyes of all rats were grossly examined at the time of necropsy using a saline-dipped, microscopic slide technique.
An interim sacrifice was performed following 11 exposures on the surviving male and female CD-1 and CF-1 mice from the 450 ppm exposure group and three control mice per sex per strain for comparison purposes. Since there were no surviving female CD-1 mice from the 450 ppm exposure group, no control female mice of this strain were sacrificed for the comparison. At this sacrifice interval, only a gross pathologic examination was performed. On a representative number of mice that died, the abdominal and thoracic cavities were exposed and the entire body was placed in 10% NEF. At the termination of exposure, all surviving mice were removed from the study and were killed. A necropsy was not performed. Mice were not subjected to histopathologic evaluation after death or at sacrifice since a more detailed mouse subchronic study was planned.
The scheduled interim sacrifice occurred for one male and three females of the 450 ppm group and for four rats per sex from the 150, 100, 50 ppm, and control groups. Tissue specimens and organ weights were obtained at this time. The final sacrifice involved five rats per sex per the 150, 100, 50 ppm, and control groups and was performed on the male rats following 36 completed exposures and on the female rats after 37 completed exposures.
Two unscheduled sacrifices of the rats were performed, prior to the interim sacrifice, because of the large numbers of mortalities noted in the 450 ppm group. This would have prevented statistical comparisons of the anatomical and clinical pathology data at the scheduled interval. The first unscheduled sacrifice was conducted after 8 completed exposures, on six female rats from the 450 ppm group and four control females. Following 12 completed exposures five males from the 450 ppm group and four control group males were sacrificed. In the event that a comparison would have been warranted between the pathologic findings of the males and females, two female rats of the 450 ppm group were also sacrificed and tissue specimens obtained at this time.
The liver, kidney, spleen, brain, heart, and lungs of all rats, and testes from all male rats, were weighed at the time of sacrifice. There were no organ weights obtained from the two female rats of the 450 ppm group which were sacrificed unscheduled. The liver, kidneys, and spleen were weighed from mice of the 450 ppm and air control groups that were sacrificed immediately following the last exposure for the 450 ppm group. [Note: This is an addition to the protocol.] All organ weights were recorded as absolute weight and as a percentage of body weight.
Statistics:
The fiducial limit of 0.05 (two-tailed) was selected as the critical level of significance. All data of each exposure group were compared statistically to the air-control group by using the following tests: Continuous variable data were analyzed by Bartlett's test for homogeneity of variance, analysis of variance and Duncan' s multiple range test. Whenever the F value for 'analysis of variance was significant and Bartlett's test indicated homogeneous variance, Duncan's multiple range test was used to denote which groups differed significantly from the control. When Bartlett's test indicated heterogeneous variance, the F-test was employed to compare each exposure group with the air-control group. The type of t-test then used was selected according to the significance of the F value. The Student's t-test was used when the F value was not significant; the Cochran t-test was used when the F value was significant. For cases in the food and water consumption measurements where one or more of the values differ from the others by a large amount and no observed cause for this could be identified but a spill was suspected, Chauvenet's criterion was applied to determine if data should be rejected. Appropriate non-parametric data were compared by employing themultiple sum of ranks test. The median and semi-interquartile range were reported for these data. Discontinuous frequency data were analyzed by using Fisher's exact test.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
Mortality
For both the rats and mice, treatment-related mortality occurred only in the 450 ppm exposure group. Because of the high incidence of mortality noted by the end of the second exposure week for the mice and the third exposure week for the rats, the exposures at this concentration for both species were terminated on November 8, 1976. No appreciable differences in mortality rates were noted between male and female rats or mice. On the basis of mortality, the CD-1 mice appeared to be slightly more affected by exposure to ethylene oxide than the CF-1 mice or the Fischer 344 rats.

Observations
Many signs of adverse effects were noted during the second and third exposure weeks for the rats and mice in the 450 ppm exposure group. These signs included red crusty material around eyes and nose, paresis or poor coordination of hind quarters, hunched posture, diarrhoea, irregular breathing, red urine (mice), convulsions, abnormal muscle contractions including tremors, piloerection, and death. During this period, some of these same signs were also noted in the rats exposed to 150 ppm of ethylene oxide. However, the severity in most cases was slight and there were no deaths nor signs convulsions or abnormal muscle contractions. These signs disappeared after the fourth exposure week. In general, there were no remarkable signs for the mice of the 150 ppm concentration group, nor for the rats and mice in the remaining two exposure groups for the entire study.

Neuromuscular Function Test (rats only):
The detailed clinical observations and neuromuscular function test were initially conducted to determine if there were a change in the results obtained on a Friday (i.e. fallowing 5 completed exposure days) and on a Monday (i.e. following subsequent exposure after 2 days of no exposure). It was found that there were no major differences noted in the observations made on these two days (18 and 19 completed exposures) for the male and female rats in the 150 ppm exposure group. During the 5, 6 and 7th exposure weeks, the same detailed evaluations were performed on the 150 ppm and control groups of rats. Because of time limitations, only a small sample size was used during these evaluations. During this time period, no appreciable differences were noted between the ethylene oxide-exposed and control groups. At the evaluation prior to the final sacrifice, the number of animals evaluated was larger. As before, no significant differences were noted.

Body weight:
For the ethylene oxide-exposed rats in comparison with the control animals, statistically significantly depressed rates of gain in body weight were noted for all exposure concentrations excluding female rats exposed to 50 ppm of ethylene oxide. Based on body weight gains of the rats, females of the 150 ppm group were more adversely affected by ethylene oxide exposure than the males; but the males in the 50 ppm group were more affected than the females. This effect on the body weight in the males of the 50 ppm group subsided as exposures continued. For the mice, significantly depressed rates of gain in body weight were also noted for all ethylene oxide-exposure groups excluding the female CF-1 mice exposed to 100 ppm of ethylene oxide. The males were more affected by exposure than the females for both strains of mice. One strain was not more adversely affected than the other.

Urinalysis (rats only):
By inspection of the data, no treatment-related effects were noted for either sex in the 150, 100 or 50 ppm exposure groups. It was noted in the male rats of the 450 ppm group that small amounts of bilirubin were detected in four out of six samples, whereas all of the samples from the control group were negative. There were not enough female rats of the 450 ppm group sampled at this time period to make a conclusion for this sex.

Hematology (rats only):
Lymphocytopenia was observed for both sexes in the highest exposure concentration only. It was noted that the white blood cell counts for the 150, 100, and 50 ppm female exposure groups at week 8 were statistically different (lower values) from the control. Since these differences were not indicative of a dose response relationship, the significance of these statistical findings are unknown. Several erythroid parameters were statistically significantly different from the controls for both male and female rats; however, in all cases the difference was only slight (i.e. less than 10%). Of these parameters, hemoglobin concentration and the accompanying hemoglobin indices of the female rats in the 150 and 100 ppm exposure groups were statistically significantly lower than the control values. Only the hemoglobin concentrations of the males from the 150 ppm group were depressed. Most of these differences were not noted until the final necropsy.

Blood Clotting Time (rats and mice):
There were no significant effects on clotting times in either rats or mice.

Clinical Chemistry (rats only):
Several individual values for many of the clinical chemistry measurements in male and female rats of the 450 ppm exposure group were outside the range of values for the controls. This was the case for cholesterol (elevated, males only), serum urea nitrogen (elevated), lactic dehydrogenase (elevated), alkaline phosphatase (decreased), hydroxybuteric dehydrogenase (elevated), serum glutamic oxalacetic transaminase (elevated, females only), serum glutamic pyruvic transaminase (elevated, females only) and cholinesterase (elevated). However, the only statistically significant differences in the group values were for cholesterol (elevated), alkaline phosphatase (depressed) and cholinesterase (elevated, males only). For the remaining ethylene oxide exposure groups, there were a few statistically significant differences noted for cholesterol, lactic dehydrogenase, alkaline phosphatase, and hydroxybuteric dehydrogenase; however, none of these differences were considered biologically significant or indicative of a treatment-related response.

Organ weight:
Because of the emaciated condition of the surviving males and females in the 450 ppm exposure group, all organ weights, except brain and kidney (females only), were appreciably depressed. Statistically significant differences were noted for the relative kidney weights (expressed as percentage of body weight) of the male and female rats in the 150 ppm exposure group. The biological significance of this finding is unknown because, for both sexes, the absolute kidney weights were similar to the control values, and for the females, the significant difference was not observed at the subsequent necropsy interval. At the final sacrifice, the relative testes weights were statistically significantly greater than the controls in all ethylene oxide-exposure groups (except the 450 ppm group which was terminated early). These statistical differences are believed to reflect the alteration in body weight rather than an effect on the organ per se, since the body weights were slightly depressed for all ethylene oxide-exposure groups, but the absolute weights of the testes from the ethylene oxide-exposure groups were similar to the controls. The differences noted were not indicative of testicular atrophy. Sporadic statistically significant differences were noted for the absolute liver and spleen weights and relative brain weight in the females at the final sacrifice; however, these differences were not considered to be either treatment related or biologically significant. Some of these differences may be explained by the lower than control body weights of these animals, particularly in the 150 and 50 ppm level, since the absolute organ weights of these animals were similar to the controls. The liver, kidney, and spleen from the mice of the 450 ppm (and air control) group were weighed at the final sacrifice of this group. It is unknown what the significance is of the alterations in organ weights at this interval since there were only limited numbers (3 to 4) of animals in most groups and because the body weight was markedly depressed.

Gross and histopathologic evaluation (rats only):
In general, exposure to 450 ppm of ethylene oxide for 8 to 13 days resulted in lesions in the mucosa of the nasal cavity, testicular degeneration, and thymic atrophy. Although there was no clear-cut pathogenic mechanism evident, the most probable cause of death was attributed to vascular damage as evidenced by gastrointestinal bleeding, urinary tract bleeding and/or pulmonary edema. Asphyxia due to nasal cavity obstruction was a less frequent cause of death. In only the male rats of the 450 ppm group was testicular degeneration with abnormal spermatocytes and atrophy of the seminiferous tubules noted. In the epididymides, both hypospermia and aspermia were noted in rats from this exposure group. No rats at the other exposure levels had any evidence of testicular degeneration nor other significant pathologic changes after 36 to 37 days of exposure.
Dose descriptor:
NOAEC
Effect level:
< 50 ppm (nominal)
Based on:
test mat.
Remarks:
in rats and mice
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
not determinable
Remarks:
no NOAEC identified
Critical effects observed:
yes
Lowest effective dose / conc.:
450 ppm (nominal)
System:
haematopoietic
Organ:
blood
Treatment related:
yes
Dose response relationship:
no
Critical effects observed:
yes
Lowest effective dose / conc.:
450 ppm
System:
nervous system
Organ:
brain
Treatment related:
yes
Dose response relationship:
yes
Endpoint:
chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
The current study was designed to investigate the mutagenic potential of EO and PO in monkeys following 2-year exposures under controlled experimental conditions.
GLP compliance:
not specified
Specific details on test material used for the study:
- Purity: 99.7%
- Physical appearance: gas
Species:
monkey
Strain:
Macaca fascicularis
Sex:
male
Details on test animals or test system and environmental conditions:
60 adult male cynomolgus monkeys were randomly assigned to 5 groups, each consisting of 12 monkeys. A shared common control group was employed. Body weight was 5.31 +/- 0.82 kg at the start of the study. The monkeys were housed in stainless-steel housing cages with automatic watering in animal rooms except during the exposure period. A 12 h on : 12 h off lighting system was maintained. Monkeys were fed a standard pellet diet of Purina, once daily at the end of the exposure day, with fresh fruit provided 2 - 3 times per week. Water was provided ad libitum except during exposures.
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Details on inhalation exposure:
Control monkeys were exposed in chambers to filtered air. 50 ppm concentrations were selected since they were the current federal workplace standards for EO, at the time the exposures were started. EO 100 ppm concentrations were chosen since they approximate maximum tolerated airborne concentrations for monkeys.
Animal exposures were conducted using 4.5-m3 stainless-steel and glass inhalation chambers. Chambers were operated under dynamic flow conditions with tangential airfeed manifolds maintained at 40 liters/min with a pressure of -0.25 cm water. Chamber airflows provided 12 - 15 air changes/hr. Temperature and humidity were maintained at 24 +/- 3°C and 50 +/- 10%, respectively. EO was vaporized using an Atinco EO vapor generator, and the vapors were metered into the filtered air intake of the chamber. Chamber concentrations were monitored 2 to 4 times per hour in an infrared analyzer. Charcoal tube samples of the test atmospheres analyzed by gas chromatography verified that the chamber concentrations were within 10% of the target values, and that the control chamber atmosphere was free of EO.
Blood collection: About 4 ml blood was collected from the femoral vein of each monkey while the animal was tranquilized following im ketamine hydrochloride. Blood was immediately placed in sterile stoppered tubes, two per animal, containing 0.1 ml sodium heparin, mixed thoroughly for at least 15 min, and stored at room temperature until cultured. Blood collections were initiated during the final month of exposure and were completed prior to the termination of the 2-year exposures.
Duration of treatment / exposure:
2 years
Frequency of treatment:
7 h/d, 5 d/w
Dose / conc.:
50 ppm
Dose / conc.:
100 ppm
No. of animals per sex per dose:
12
Control animals:
yes
Details on results:
Monkeys, with the exception of the EO 100 ppm group, tolerated the exposures well. The EO 100 ppm group had a statistically significant reduced mean body weight compared to the control group beginning at Week 19 and continuing through Week 104. Five monkeys died during the 2-year exposures, one each in the EO 50 and EO 100 ppm groups. These deaths did not appear to be related to oxide exposure. In addition, 10 monkeys, 2 per group, were killed at the termination of exposures. The remaining 44 monkeys are still being maintained and are being examined quarterly for body weight and hematological changes.
Dose descriptor:
NOAEC
Effect level:
< 50 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable
Remarks:
no NOAEC identified
Critical effects observed:
no
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
18 mg/m³
Study duration:
chronic
Species:
rat
Quality of whole database:
Scientifically acceptable data available.
System:
central nervous system
Organ:
other: central nervous system

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
exposure duration 7 to 8 weeks
GLP compliance:
yes (incl. QA statement)
Remarks:
testing lab.
Limit test:
no
Specific details on test material used for the study:
- Supplier: Union Carbide Corporation
- Physical appearance: liquid
- Purity: > 99.9%
Species:
other: rats and mice
Strain:
other: Fischer 344 (rats), CD1 and CF1 (mice)
Sex:
male/female
Details on test animals or test system and environmental conditions:
Male and female rats were obtained from Microbiological Associates, Inc. (Walkersville, MID) and male and female mice were obtained from Charles River Breeding Laboratories, Wilmington, MA and Saint Constance, Ontario, Canada, respectively.
The Fischer 344 rat strain was selected because of its intended use in a two-year ethylene oxide vapor study. The reasons for selection of this strain included (i) fact that this strain has been accepted for the carcinogen bioassay program of the National Toxicology Program, (ii) it had been widely accepted in chronic inhalation studies partially due to its small size which lessen the heat load in the inhalation chamber, and (iii) because of its good longevity record in chronic studies. Two strains of mice were selected to determine if there were any appreciable differences in the toxic responses between strains of mice. At the laboratory, both strains have been used in previous toxicology studies.
All animals were approximately 4 weeks old upon arrival at the laboratory and appeared in good health. Quality control examinations which included body weight determinations, fecal examination for intestinal parasites by zinc sulfate flotation, evaluation of the nasal larynx and lung for aerobic bacteriologic flora, and micropathological examination of selected tissues were performed on a portion of the rats upon receipt. The eyes of all rats were grossly examined (visual examination) for lesions prior to the start of the exposures. The results of the quality control and microscopic examinations were within normal limits for commercially available, specific pathogen-free rats; therefore, the animals were determined to be of suitable quality for the inhalation study. Only limited quality control evaluations were performed on the mice since the main objective of this study was to evaluate the effects of ethylene oxide vapor exposure on rats.
All animals were identified with individual numbers by a sequential toe-clipping procedure. Listed on each color-coded cage card (identifying exposure concentration) was the identification numbers of the animals within the cage.
The animals were observed for two 2eeks prior to assignment into exposure groups. During this period, all animals were weighed three to four times and assigned to exposure levels using a card-based randomization system. An animal was not used for group assignment if it were not gaining weight
normally, if abnormal clinical observations were noted, or if the body weight at the time of group assignment were above or below to standard deviations from the mean of all animals of that sex.
The rats were housed five per cage and mice were housed three per cage in suspended, stainless steel, wire mesh cages, 35 cm x 37 cm x 18 cm high for rats and 35 cm x 17 cm x 18 cm high for mice (separated by test group and sex). The animals were housed in the same cages during exposure and non-exposure periods. When the animals were in the room, the temperature and humidity controlling devices of the non-recirculated air supply to the room were set to maintain the environment between 66 and 77 °F and 30 to 70% relative humidity. The fluorescent lighting was set on a 12-hour photoperiod. Water was available, ad libitum, to the rats and mice throughout the exposure and non-exposure periods by an automatic watering system. Purine diet was available ad libitum throughout the non-exposure period only.
A stainless steel shelf pan was placed between each level of cages to prevent urinary and fecal contamination of the animals on lower tiers. These pans were in place during the exposures. After each exposure, clean pans with absorbent paperboard were placed on the carriers. Before exposure, the absorbent paperboard was removed.
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
The animals were exposed to ethylene oxide vapor or room air in 3800-liter stainless steel-lined inhalation chambers. Each chamber had glass windows for observation of animals. Chamber temperature and relative humidity were recorded three times a day on average. The airflow through the chambers was maintained at approximately 1000 liters per minute.
Liquid aliquots (approximately 5 pounds) from the storage drum were transferred to a stainless steel cylinder. This cylinder which was attached to the generation system was maintained at approximately 30°C by mneans of a constant temperature recirculating bath. The ethylene oxide vapor pressure generated at that temperature was utilized to conduct the gas through stainless steel tubing and a pressure-reducing regulator to maintain approximately three psi internal pressure. Manifolds of stainless steel tubing directed the vapor through control values and flowmeters to the chamber air-inlet duct. Ethylene oxide vapor was diluted at the inlet duct with exposure room air and drawn into the inhalation exposure chamber.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All chambers were monitored for ethylene oxide concentration by means of a Varian 2700 gas chromatograpgh (GC). The chamber atmosphere samples were manually injected into the GC and an automatic recording system connected to the GC transferred the data to a printer and onto magnetic trap. A known primary standard concentration of ethylene oxide was sampled several times before the start of daily chamber analysis to check the monitoring system. Each day, approx. four samples were analysed from each ethylene oxide-exposure chamber; and between one and three samples of the air within the exposure room and a similar number of samples from the control chamber were analysed.
Duration of treatment / exposure:
7 - 8 weeks
Frequency of treatment:
6 h/d, 5 d/w
Dose / conc.:
50 ppm (nominal)
Remarks:
90 mg/m³
Dose / conc.:
100 ppm (nominal)
Remarks:
180 mg/m³
Dose / conc.:
150 ppm (nominal)
Remarks:
270 mg/m³
Dose / conc.:
450 ppm (nominal)
Remarks:
810 mg/m³
No. of animals per sex per dose:
Each of the groups contained 35 male and 35 female Fischer 344 rats und 15 male and 15 female CD-1 und the same number of CF-1 mice, with the exception of the control group which contained an additional 10 male and 10 female rats. Included in this number of rats were "extra" rats for each group. The ethylene oxide exposure groups contained 7/sex and the control group contained 17/sex. These rats were to be used if an unscheduled necropsy interval was necessary. If these additional rats were not used, they were sacrificed and removed from the study at the termination of the exposures for that group. Necropsy was not performed for these rats.
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Target concentrations of 450, 150, 100, and 50 ppm of ethylene oxide were selected for the exposure concentrations based on results of previous rat and mouse studies as reported by Hollingsworth, et al. (1956) and Jacobson, et al. (1956). All animals were exposed for six hours per day, five days per week; the male rats received 36 exposures, female rats received 37 exposures, and both sexes and strains of mice received 33 exposures. The rats had only four exposures during the first week. Control (air-exposed) animals were handled in an identical manner as the ethylene oxide-treated animals. Animal cages were rotated within each chamber on a weekly basis to compensate for any possible, but undetected, variation in chamber exposure conditions (i.e., concentration, temperature, and/or relative humidity). Because of high mortality, exposures for the 450 ppm group were terminated, following 14 exposures for rats and 11 exposures for mice.
Observations and examinations performed and frequency:
During the six-hour exposure, a portion of the animals were observed several times through the chamber windows. However, most changes in general health status were better observed for all animals immediately before and after the exposure. Any abnormalities in appearance were recorded at these times.
A neuro-muscular function examination was performed on the male and female rats of the 150 ppm and control groups following 18, 19, 21, 23, 26, 28, 31, 33, and 37 completed exposures. [Note: This is an addition to the protocol]. The same five rats per sex per group were evaluated at each examination period, with the exception of the final examination where 12 female rats per group and 7 males from the 150 ppm group and 8 control group males were examined. The examination that occurred following 33 completed exposures only consisted of examining the righting reflex and climbing ability of the rate.
All animals were weighed the morning before the first exposure. This weight, termed the pre-exposure reference weight, was subtracted from each subsequent weight determination to obtain the change in body weight. Body weights were measured weekly throughout the study, rats an Friday and mice an Tuesday. In addition, all animals were weighed at the time of sacrifice.
Urine (rats only) was collected for approximately two hours from both male and female rats prior to the interim and final sacrifice periods. The urine was semi-quantitatively analysed by a dip stick method. The items evaluated were as follows: bilirubin, glucose, ketones, nitrite, occult blood, pH, protein, and urobilinogen. Specific gravity was determined by using a refractometer. Urine volumes were not measured.
Hematologic and serum clinical chemistry evaluations (rats only) were performed on randomly-selected male and female rats at three and eight weeks of exposure, and on two days preceding each sacrifice period. At an unscheduled sacrifice during the second exposure week, only blood from female rats of the 450 ppm and control groups were submitted for evaluation.
All rats had free access to food and water prior to evaluation but were deprived of both food and water during the bleeding period. The rats were lightly anesthetized with methoxyflurane and blood was collected from the interior vena cava. After blood collection, the animals were sacrificed by cervical dislocation.
Other than the samples used for clotting time determinations, all samples for hematology were collected in heparinized tubes. Hematologic parameters included white blood cell count , white blood cell differential count, red blood cell count, packed red cell volume, hemoglobin concentration, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration and clotting time. Clotting was determined by drawing a needle through a fresh drop of blood until a strand was noted. Clotting time was also determined for the male CD-1 and CF-1 and female CF-1 mice following 10 exposures (450 ppm and control only) and 33 exposures (150 ppm and control only). The CD-1 female mice of the 450 ppm exposure group were all dead by the tenth exposure, consequently, clotting times for the CD-1 female mice of the control group were not determined.
The biochemical analysis included cholesterol, serum urea nitrogen, creatinine, lactic dehydrogenase, alkaline phosphatase, albumin, creatine phosphokinase, alpha-hydroxybutyric dehydrogenase, serum glutamic oxaloacetie transaminase, serum glutamic pyruvic transaminase, calcium, glucose, cholinesterase, total bilirubin, total protein, and glutamyl transpeptidase.
Sacrifice and pathology:
At each sacrifice period, the rats and mice were killed by severing the cervical spinal cord and exsanguination via the jugular und carotid vessels. For all rats, the sciatic nerve was dissected free und fixed in 3% phosphate buffered glutaraldehyde for possible future examination. The femur was removed and a fresh bone marrow impression smear was prepared and stained with Wright-Giemsa stain for each rat. All viscera from these animals were removed and representative samples were fixed in 10% neutral buffered formalin. The lungs were weighed and then inflated with 10% NBF. The eyes of all rats were grossly examined at the time of necropsy using a saline-dipped, microscopic slide technique.
An interim sacrifice was performed following 11 exposures on the surviving male and female CD-1 and CF-1 mice from the 450 ppm exposure group and three control mice per sex per strain for comparison purposes. Since there were no surviving female CD-1 mice from the 450 ppm exposure group, no control female mice of this strain were sacrificed for the comparison. At this sacrifice interval, only a gross pathologic examination was performed. On a representative number of mice that died, the abdominal and thoracic cavities were exposed and the entire body was placed in 10% NEF. At the termination of exposure, all surviving mice were removed from the study and were killed. A necropsy was not performed. Mice were not subjected to histopathologic evaluation after death or at sacrifice since a more detailed mouse subchronic study was planned.
The scheduled interim sacrifice occurred for one male and three females of the 450 ppm group and for four rats per sex from the 150, 100, 50 ppm, and control groups. Tissue specimens and organ weights were obtained at this time. The final sacrifice involved five rats per sex per the 150, 100, 50 ppm, and control groups and was performed on the male rats following 36 completed exposures and on the female rats after 37 completed exposures.
Two unscheduled sacrifices of the rats were performed, prior to the interim sacrifice, because of the large numbers of mortalities noted in the 450 ppm group. This would have prevented statistical comparisons of the anatomical and clinical pathology data at the scheduled interval. The first unscheduled sacrifice was conducted after 8 completed exposures, on six female rats from the 450 ppm group and four control females. Following 12 completed exposures five males from the 450 ppm group and four control group males were sacrificed. In the event that a comparison would have been warranted between the pathologic findings of the males and females, two female rats of the 450 ppm group were also sacrificed and tissue specimens obtained at this time.
The liver, kidney, spleen, brain, heart, and lungs of all rats, and testes from all male rats, were weighed at the time of sacrifice. There were no organ weights obtained from the two female rats of the 450 ppm group which were sacrificed unscheduled. The liver, kidneys, and spleen were weighed from mice of the 450 ppm and air control groups that were sacrificed immediately following the last exposure for the 450 ppm group. [Note: This is an addition to the protocol.] All organ weights were recorded as absolute weight and as a percentage of body weight.
Statistics:
The fiducial limit of 0.05 (two-tailed) was selected as the critical level of significance. All data of each exposure group were compared statistically to the air-control group by using the following tests: Continuous variable data were analyzed by Bartlett's test for homogeneity of variance, analysis of variance and Duncan' s multiple range test. Whenever the F value for 'analysis of variance was significant and Bartlett's test indicated homogeneous variance, Duncan's multiple range test was used to denote which groups differed significantly from the control. When Bartlett's test indicated heterogeneous variance, the F-test was employed to compare each exposure group with the air-control group. The type of t-test then used was selected according to the significance of the F value. The Student's t-test was used when the F value was not significant; the Cochran t-test was used when the F value was significant. For cases in the food and water consumption measurements where one or more of the values differ from the others by a large amount and no observed cause for this could be identified but a spill was suspected, Chauvenet's criterion was applied to determine if data should be rejected. Appropriate non-parametric data were compared by employing themultiple sum of ranks test. The median and semi-interquartile range were reported for these data. Discontinuous frequency data were analyzed by using Fisher's exact test.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
Mortality
For both the rats and mice, treatment-related mortality occurred only in the 450 ppm exposure group. Because of the high incidence of mortality noted by the end of the second exposure week for the mice and the third exposure week for the rats, the exposures at this concentration for both species were terminated on November 8, 1976. No appreciable differences in mortality rates were noted between male and female rats or mice. On the basis of mortality, the CD-1 mice appeared to be slightly more affected by exposure to ethylene oxide than the CF-1 mice or the Fischer 344 rats.

Observations
Many signs of adverse effects were noted during the second and third exposure weeks for the rats and mice in the 450 ppm exposure group. These signs included red crusty material around eyes and nose, paresis or poor coordination of hind quarters, hunched posture, diarrhoea, irregular breathing, red urine (mice), convulsions, abnormal muscle contractions including tremors, piloerection, and death. During this period, some of these same signs were also noted in the rats exposed to 150 ppm of ethylene oxide. However, the severity in most cases was slight and there were no deaths nor signs convulsions or abnormal muscle contractions. These signs disappeared after the fourth exposure week. In general, there were no remarkable signs for the mice of the 150 ppm concentration group, nor for the rats and mice in the remaining two exposure groups for the entire study.

Neuromuscular Function Test (rats only):
The detailed clinical observations and neuromuscular function test were initially conducted to determine if there were a change in the results obtained on a Friday (i.e. fallowing 5 completed exposure days) and on a Monday (i.e. following subsequent exposure after 2 days of no exposure). It was found that there were no major differences noted in the observations made on these two days (18 and 19 completed exposures) for the male and female rats in the 150 ppm exposure group. During the 5, 6 and 7th exposure weeks, the same detailed evaluations were performed on the 150 ppm and control groups of rats. Because of time limitations, only a small sample size was used during these evaluations. During this time period, no appreciable differences were noted between the ethylene oxide-exposed and control groups. At the evaluation prior to the final sacrifice, the number of animals evaluated was larger. As before, no significant differences were noted.

Body weight:
For the ethylene oxide-exposed rats in comparison with the control animals, statistically significantly depressed rates of gain in body weight were noted for all exposure concentrations excluding female rats exposed to 50 ppm of ethylene oxide. Based on body weight gains of the rats, females of the 150 ppm group were more adversely affected by ethylene oxide exposure than the males; but the males in the 50 ppm group were more affected than the females. This effect on the body weight in the males of the 50 ppm group subsided as exposures continued. For the mice, significantly depressed rates of gain in body weight were also noted for all ethylene oxide-exposure groups excluding the female CF-1 mice exposed to 100 ppm of ethylene oxide. The males were more affected by exposure than the females for both strains of mice. One strain was not more adversely affected than the other.

Urinalysis (rats only):
By inspection of the data, no treatment-related effects were noted for either sex in the 150, 100 or 50 ppm exposure groups. It was noted in the male rats of the 450 ppm group that small amounts of bilirubin were detected in four out of six samples, whereas all of the samples from the control group were negative. There were not enough female rats of the 450 ppm group sampled at this time period to make a conclusion for this sex.

Hematology (rats only):
Lymphocytopenia was observed for both sexes in the highest exposure concentration only. It was noted that the white blood cell counts for the 150, 100, and 50 ppm female exposure groups at week 8 were statistically different (lower values) from the control. Since these differences were not indicative of a dose response relationship, the significance of these statistical findings are unknown. Several erythroid parameters were statistically significantly different from the controls for both male and female rats; however, in all cases the difference was only slight (i.e. less than 10%). Of these parameters, hemoglobin concentration and the accompanying hemoglobin indices of the female rats in the 150 and 100 ppm exposure groups were statistically significantly lower than the control values. Only the hemoglobin concentrations of the males from the 150 ppm group were depressed. Most of these differences were not noted until the final necropsy.

Blood Clotting Time (rats and mice):
There were no significant effects on clotting times in either rats or mice.

Clinical Chemistry (rats only):
Several individual values for many of the clinical chemistry measurements in male and female rats of the 450 ppm exposure group were outside the range of values for the controls. This was the case for cholesterol (elevated, males only), serum urea nitrogen (elevated), lactic dehydrogenase (elevated), alkaline phosphatase (decreased), hydroxybuteric dehydrogenase (elevated), serum glutamic oxalacetic transaminase (elevated, females only), serum glutamic pyruvic transaminase (elevated, females only) and cholinesterase (elevated). However, the only statistically significant differences in the group values were for cholesterol (elevated), alkaline phosphatase (depressed) and cholinesterase (elevated, males only). For the remaining ethylene oxide exposure groups, there were a few statistically significant differences noted for cholesterol, lactic dehydrogenase, alkaline phosphatase, and hydroxybuteric dehydrogenase; however, none of these differences were considered biologically significant or indicative of a treatment-related response.

Organ weight:
Because of the emaciated condition of the surviving males and females in the 450 ppm exposure group, all organ weights, except brain and kidney (females only), were appreciably depressed. Statistically significant differences were noted for the relative kidney weights (expressed as percentage of body weight) of the male and female rats in the 150 ppm exposure group. The biological significance of this finding is unknown because, for both sexes, the absolute kidney weights were similar to the control values, and for the females, the significant difference was not observed at the subsequent necropsy interval. At the final sacrifice, the relative testes weights were statistically significantly greater than the controls in all ethylene oxide-exposure groups (except the 450 ppm group which was terminated early). These statistical differences are believed to reflect the alteration in body weight rather than an effect on the organ per se, since the body weights were slightly depressed for all ethylene oxide-exposure groups, but the absolute weights of the testes from the ethylene oxide-exposure groups were similar to the controls. The differences noted were not indicative of testicular atrophy. Sporadic statistically significant differences were noted for the absolute liver and spleen weights and relative brain weight in the females at the final sacrifice; however, these differences were not considered to be either treatment related or biologically significant. Some of these differences may be explained by the lower than control body weights of these animals, particularly in the 150 and 50 ppm level, since the absolute organ weights of these animals were similar to the controls. The liver, kidney, and spleen from the mice of the 450 ppm (and air control) group were weighed at the final sacrifice of this group. It is unknown what the significance is of the alterations in organ weights at this interval since there were only limited numbers (3 to 4) of animals in most groups and because the body weight was markedly depressed.

Gross and histopathologic evaluation (rats only):
In general, exposure to 450 ppm of ethylene oxide for 8 to 13 days resulted in lesions in the mucosa of the nasal cavity, testicular degeneration, and thymic atrophy. Although there was no clear-cut pathogenic mechanism evident, the most probable cause of death was attributed to vascular damage as evidenced by gastrointestinal bleeding, urinary tract bleeding and/or pulmonary edema. Asphyxia due to nasal cavity obstruction was a less frequent cause of death. In only the male rats of the 450 ppm group was testicular degeneration with abnormal spermatocytes and atrophy of the seminiferous tubules noted. In the epididymides, both hypospermia and aspermia were noted in rats from this exposure group. No rats at the other exposure levels had any evidence of testicular degeneration nor other significant pathologic changes after 36 to 37 days of exposure.
Dose descriptor:
NOAEC
Effect level:
< 50 ppm (nominal)
Based on:
test mat.
Remarks:
in rats and mice
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
not determinable
Remarks:
no NOAEC identified
Critical effects observed:
yes
Lowest effective dose / conc.:
450 ppm (nominal)
System:
haematopoietic
Organ:
blood
Treatment related:
yes
Dose response relationship:
no
Critical effects observed:
yes
Lowest effective dose / conc.:
450 ppm
System:
nervous system
Organ:
brain
Treatment related:
yes
Dose response relationship:
yes
Endpoint conclusion
Endpoint conclusion:
adverse effect observed

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The following is quoted from the RAC opinion proposing a harmonized classification and labelling at the EU level of ethylene oxide:

"In the case of ethylene oxide, there are animal data showing clear effects on peripheral nervous system. In most of the cases, effects have been observed in animals in long term studies (1 to

2 years) at exposures of 200 ppm or higher. Mandella (1997b) reported decreased hindlimb grip strengths at 300 ppm already after 4 weeks of exposure. In the long term study (14 weeks),

Mandella et al. (1997c) reported similar findings at 200 ppm, but not at 100 ppm. Lynch (1984a) reported myopathy in rats after exposure to 100 ppm for 2 years, and Snellings et al. (1984) a

dose-related reduction in locomotor function and abnormal posture starting from 50 ppm in a subchronic (10-11 weeks) study. If only animal data on the neurotoxicity of ethylene oxide is

taken into account, the weight of evidence supports STOT RE 2 classification for neurotoxicity. However, the similar effects on the PNS has been reported also in several human case reports.

These reports describe not only neurotoxic symptoms in humans, but also measured effects on nerve conduction velocities indicative of sensorimotor neuropathy, and axonal degeneration

observed in nerve biopsies of exposed workers. Controlled studies in ETO inhalation exposed workers are limited in size and/or methodology but supported the signs and symptoms reported

in case reports. RAC is in the opinion that the human evidence together with data from experimental animals provides sufficient evidence to support the classification of ethylene oxide

as STOT RE 1 for neurotoxicity. Regarding haematotoxicity, in animals ethylene oxide caused effects on blood parameters at subchronic (13 weeks) exposures to 255-500 ppm. However, the effects even at 500 ppm were rather moderate and decreases were seen only up to 10-14% in haemoglobin, RBC or haematocrit levels. The data from humans was limited and variable. According to the CLP criteria, the substance can be classified for STOT RE if there are any consistent and significant adverse effect in clinical biochemistry, haematology or urinalysis. In the case of blood parameters, reduction in Hb ≥ 20% is considered as a significant adverse effect. When taking into account that in the case of ethylene oxide the reductions in haemoglobin levels stayed well below 20%

even at doses exceeding the guidance range of 50-250 ppm for STOT RE 2, no classification on the basis of haematological effects is proposed."

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

In accordance with the Prosal by RAC issued in 2017, ethylene oxide is classified as STOT RE 1; H372 for effects on the nervous system.