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Repeated dose toxicity: inhalation

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1982
Report date:
1982

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethylene oxide
EC Number:
200-849-9
EC Name:
Ethylene oxide
Cas Number:
75-21-8
Molecular formula:
C2H4O
IUPAC Name:
oxirane
Specific details on test material used for the study:
- Supplier: Union Carbide Corporation
- Physical appearance: liquid
- Purity: > 99.9%

Test animals

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.

Administration / exposure

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
Doses / concentrationsopen allclose all
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.

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

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

Target system / organ toxicity

open allclose all
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

Applicant's summary and conclusion

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