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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:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Inhalation Toxicity of Butylene Oxide.
Author:
Miller, R.R., Quast, J.R., Ayres, J.A., McKenna, M.J.
Year:
1981
Bibliographic source:
Fund. Appl. Toxicol. 1, 319-324
Reference Type:
study report
Title:
Unnamed
Year:
1980
Report date:
1980

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1,2-epoxybutane
EC Number:
203-438-2
EC Name:
1,2-epoxybutane
Cas Number:
106-88-7
Molecular formula:
C4H8O
IUPAC Name:
oxolane
Test material form:
liquid

Test animals

Species:
rat
Strain:
Fischer 344
Details on species / strain selection:
Selection of the stain was based on a variety of considerations including hardiness, life-span, spontaneous respiratory disease incidence, spontaneous tumor incidence, genetic stability, availability, historical background data, and the extent to which these species and strains are utilized for chronic toxicity/carcinogenicity studies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories,Inc., Portage, MI
- Age at study initiation: 4-6 weeks of age
- Weight at study initiation: no data
- Housing: housed in stainless steel cages with wire bottoms, two per cage at all times
- Diet: ad libitum, standard laboratory diet (Purina Laboratory Chow, Ralston Purina Co., St. Louis, MO)
- Water: ad libitum
- Acclimation period: 10-14 days

DETAILS OF FOOD AND WATER QUALITY: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 21
- Humidity (%): 55
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
One cubic meter stainless steel and glass Rochester-type inhalation chambers were used for animal exposures. Temperature and relative humidity in the chambers and in the rooms were controlled by a system designed to maintain temperature at approximately 70°F and relative humidity at approximately 50%. Temperature and relative humidity were operated under dynamic airflow conditions at a slight negative pressure relative to the surrounding area.
Exposure concentrations of butylene oxide were generated by metering the liquid test substance at calculated rates into glass vaporization tubes as described by Miller et al . (1980b). Vapors from the tubes were swept into the chamber inlet ducts with compressed air where there was further mixing and dilution with incoming air. The compressed air was preheated with a compressed air flameless heat torch (Master, Model FHT-4) to facilitate complete vaporization of the liquid test substance. Total chamber airflow was maintained at approximately 200 liters per minute.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The nominal concentration (ratio of the amount of butylene oxide vaporized to the total amount of air through the chamber) was calculated for each
chamber on a daily basis. The actual concentration of butylene oxide in each chamber was measured 5-6 times per exposure day by infrared spectroscopy using a MIRAN I infrared gas analyzer (Wilks/Foxboro Inc., Norwalk, CT) at a wavelength of 11 u. The daily time-weighted average (TWA) analytical concentration was calculated for each exposure chamber on a daily basis.
Duration of treatment / exposure:
6 hours/day, 5 days/week
Frequency of treatment:
A predesignated group of 5 animals of each sex per exposure level was sacrificed after 30 days (24 exposures); the remaining animals were sacrificed after 93 days on study (66 exposures).
Doses / concentrationsopen allclose all
Dose / conc.:
75 ppm (nominal)
Remarks:
0, 74.0 +/- 2.4 ppm daily time-weighted average (TWA) analytical concentration
(0.22 mg/L target concentration)
Dose / conc.:
150 ppm (nominal)
Remarks:
148.8 +/- 4.9 ppm daily time-weighted average (TWA) analytical concentration
(0.44 mg/L target concentration)
Dose / conc.:
600 ppm (nominal)
Remarks:
601.6 +/- 8.2 ppm daily time-weighted average (TWA) analytical concentration
(1.77 mg/L target concentration)
No. of animals per sex per dose:
15
Control animals:
yes
Details on study design:
Rats were exposed via inhalation to 0, 75, 150 or 600 ppm test item vapors (0.22, 0.44 or 1.77 mg/L) 6 hours per day, 5 days per week (excluding holidays) for a total of 13 weeks in order to assess the subchronic toxicologic effects. All animals were observed daily for signs of toxicity and changes in appearance or demeanor. Body weights were recorded. Hematology, clinical chemistry, urinalysis, gross necropsy and histopathology was conducted. Additional animals for a concurrent study, which were purchased from the supplier at the same time as animals for the 90-day study, were placed in the chambers. These additional animals were predesignated for studies involving blood level measurements of the test material and hepatic glutathione measurements, as indicated in a separate protocol.
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
All animals were observed daily for signs of toxicity and changes in appearance or behaviour.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded immediately prior to the first exposure and weekly thereafter. In order to detect subtle effects on growth, body weight gains were calculated by subtracting the day zero body weight of each animal from its weight during the exposure interval. Final body weights were recorded immediately prior to sacrifice.

FOOD EFFICIENCY: No

OPHTHALMOSCOPIC EXAMINATION: Yes

HAEMATOLOGY: Yes
Packed Cell Volume, Hemoglobin, Erythrocyte Count and Morphology, Total Leukocyte and Differential Leukocyte Counts
Rat blood samples were taken by tail vein puncture approximately 1 week prior to scheduled sacrifice.
Haematology was conducted on 5 animals of each sex per exposure level after 30 days, and on 10 animals of each sex per exposure level after approximately 83 days.

CLINICAL CHEMISTRY: Yes
Blood Urea Nitrogen, SGPT, Alkaline Phosphatase, Glucose
Rat blood samples were taken from severed cervical blood vessels at sacrifice from animals fasted overnight. All clinical chemistry determinations were performed on serum after allowing the blood samples to clot.
Clinical chemistry was conducted on 5 animals of each sex per exposure level after 30 days, and on 10 animals of each sex per species per exposure level after approximately 83 days.

URINALYSIS: Yes
Bilirubin, pH, Glucose, Protein, Ketone, Specific Gravity (refractive index), Occult Blood, Urobilinogen
Urine samples were collected from rats approximately 1 week prior to sacrifice at the same time blood was collected for hematology.
Urinalysis was conducted on 5 animals of each sex per exposure level after 30 days, and on 10 animals of each sex per species per exposure level after approximately 83 days.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): No

LUNG BURDEN: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All surviving animals were submitted for necropsy approximately 18 hours after final exposure. Rats were deprived of food overnight prior to necropsy. Each rat was anesthetized with methoxyflurane and then decapitated after clamping the trachea. Each animal was examined internally and externally for gross pathologic alterations by a veterinary pathologist. Lungs and trachea were removed as a unit ; the lungs were then expanded to approximately their normal inspiratory volume with buffered 10% formalin . The nasal turbinates were flushed with buffered 10% formalin to improve fixation prior to placing the entire head in formalin fixative. The heart, liver, kidneys, brain, thymus and testes (males) were removed from each animal and weighed. Eyes of all rodents were examined grossly by a microscope slide technique under fluorescent illumination. Representative portions of the organs and tissues were taken from each animal and preserved in buffered 10% formalin.

HISTOPATHOLOGY: Yes
Histopathologic observations were performed on an extensive list of tissues, as well as for any grossly visible lesions, for all animals in the control and high exposure groups sacrificed after 90 days. Selected tissues suggestive of a target organ identified in the high exposure group were also examined in the middle and low exposure groups. Tissues to be examined histopathologically were processed by conventional techniques, stained with hematoxylin and eosin and evaluated by light microscopy.
Statistics:
Body weight data, organ weights, organ-to-body weight ratios, hematological values and clinical chemistry values were evaluated by analysis of variance; differences between treatment group means and control means were delineated by Dunnett's test (Steel and Torrie, 1960) if the F ratio for ANOVA was significant. Variances of group body weights were analyzed by Bartlett's test (Snedecor and Cochran, 1967). The level of significance chosen in all cases was p<0.05.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
All rats appeared normal and healthy throughout the study.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Growth of female rats in the 600 ppm group was retarded, as indicated by the statistically significant depressions in the mean body weight gains during the last few weeks of exposure. Similarly, the mean body weight gains for male rats in the 600 ppm group tended to be lower than for controls, although the mean body weight gains of these groups were not statistically different from controls at the end of the 90-day exposure interval .
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
30-Day Interim Sacrifice: The mean red blood cell count for female rats in the 600 ppm group was slightly lower than for controls. However, the PCV and Hgb values in this group were normal. In addition, the mean red cell count of 600 ppm female rats was not different from historical control values for female rats of the same strain and age. Therefore the effect on the mean red cell count of 600 ppm female rats was not considered to be treatment-related.
90-Day Sacrifice: There were no statistical differences and no apparent effects on hematologic parameters of male rats after 90 days of exposure. The mean hemoglobin values of female rats in the 150 and 600 ppm groups were slightly higher than for controls. Although statistically different from the concurrent control group, these mean hemoglobin values were within the range of normal values for female rats of the same strain and age, and there was no clear dose-response relationship. Therefore the elevated hemoglobin values in treated female rats were considered to be of negligible toxicologic significance. The mean red blood cell count of 150 ppm female rats was also statistically higher than for controls; this was considered to be a sporadic statistical difference unrelated to exposure in view of the absence of an effect on red cell counts of female rats in the 600 ppm group.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
30-Day Interim Sacrifice: There were no statistical differences or apparent effects on clinical chemistry parameters of male or female rats after 30 days of exposure.
90-Day Sacrifice: Although the mean blood urea nitrogen value (BUN) of male rats in the 600 ppm group was statistically higher than for controls after 90 days of exposure, it was within the range of normal biologic variability and not considered to be treatment related. There were no other statistical differences or apparent effects on clinical chemistry parameters of male or female rats. Historical control values were obtained from control group animals in five recently conducted inhalation studies of similar design.
Endocrine findings:
not examined
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
30-Day Interim Sacrifice . The mean urinary specific gravity values of treated male and female rats were not significantly different from controls, and there were no other apparent effects on urinary parameters of either male or female rats after 30 days of exposure.
90-Day Sacrifice. The mean urinary specific gravity values of 600 ppm male and female rats were statistically lower than for controls after 90-days of exposure; urine volume was not measured. However, there were no apparent effects on the other urinary parameters of male or female rats, nor any other indications of nephrotoxicity in either rats or mice.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
30-Day Interim Sacrifice: There were no changes in absolute or relative organ weights of rats after 30 days of exposure to butylene oxide vapors which were considered to be direct effects of the test material. All statistical differences in absolute and relative organ weights of rats were sporadic in nature and lacked a dose-response relationship, or were reflections of depressed body weight gain (e.g., the liver, kidney, heart, and relative brain weights of 600 ppm female rats).
90-Day Sacrifice: None of the mean absolute or relative organ weights of treatment groups of male rats were statistically different from controls. The mean liver, kidney, and thymus weights of female rats in the 600 ppm group were statistically different from controls. All of these organ weight changes in female rats were considered to be reflections of reduced body weight gain or stress rather than direct toxic effects of the test material.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
30 Day Interim Sacrifice: The body size of animals in the 600 ppm group was decreased, and there was decreased intra-abdominal fat as well as a decrease in the size of the thymus and mediastinal fat in these rats. Treatment related changes were not detected in rats in the 75 and 150 ppm groups after 30 days of exposure.
90-Day Sacrifice: Some male and some female rats in the 600 ppm group had decreased amounts of abdominal adipose tissue, as well as a decrease in the size of the thymus and mediastinal fat. All other gross pathologic observations in treatment groups of rats were considered to be spontaneous in nature and unrelated to exposure to the test material. No treatment related changes were detected in rats in the 75 and 150 ppm groups after 90 days of exposure.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
Histopathologic examinations of tissues from rats after 90 days of exposure revealed changes in the nasal mucosa of 600 ppm animals which were attributed to primary upper respiratory irritation produced by exposure to the test material. The microscopic changes in the nasal turbinates were minimal and were characterized by flattening of the olfactory and respiratory epithelium with some focal thickening of the respiratory epithelium. In addition, increased numbers of inflammatory cells were present in the nasal mucosa and within the lumen of the nasal cavity. Lower portions of the respiratory tract were apparently unaffected by exposure to butylene oxide vapors, although there were some observations in the lungs and trachea of a few treated female rats which were considered spontaneous in nature and unrelated to exposure. There were several microscopic changes in 600 ppm rats which were considered to be indirect effects of exposure to the test material, including decreased hepatocellular size, decreased cell content in the cortex of the thymus gland, and myeloid hyperplasia in vertebral bone marrow. All other microscopic observations were considered to be spontaneous in nature and unrelated to exposure. Notably, there were no microscopic changes suggesting nephrotoxicity which would be correlated with the specific gravity changes in 600 ppm rats. There were no microscopic observations in either male or female rats in the 75 and 150 ppm groups which were considered to be related to exposure to butylene oxide. Since only minor pathologic treatment-related effects were detected microscopically after 90 days of exposure, microscopic examinations were not performed on tissues from rats sacrificed after 30 days of exposure.
Details on results:
Exposure Conditions.
The mean daily time-weighted average (TWA) analytical concentrations and mean daily nominal concentrations of the test material were very close to the intended target concentrations for each exposure chamber. The close agreement between the mean daily TWA analytical concentration and mean daily nominal concentration for each chamber indicates that test material losses were minimal in the vapor generating and exposure systems.

Effect levels

Key result
Dose descriptor:
NOAEC
Effect level:
150 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion