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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study was conducted prior to GLP and test guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1980
Report Date:
1980

Materials and methods

Test guideline
Qualifier:
no guideline available
GLP compliance:
no
Remarks:
But the study was audited by QAU
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 1,2- Butylene oxide
- Physical state: Clear, colorless, highly flammable liquid
Lot #35 (<0.01% water, 0.003 acidity, <25 ppm aldehyde and 19 ppm chlorine and #36 (<0.01% water, 0.002 acidity, <25 ppm aldehyde and 42 ppm chlorine)

Test animals

Species:
other: Rats and Mice
Strain:
other: Fischer 344 rats and B6C3F1 mice
Sex:
male/female
Details on test animals and environmental conditions:
Twenty weanling Fischer 344 rats per sex (35 days of age) and twenty B6C3F1 mice per sex (28-42 days of age) were purchased from Charles River Breeding Laboratories, Inc., Portage, MI. Five rats per sex and five mice per sex were randomly assigned to control and each of three exposure groups using a computer-generated random number table (GRAND. CLIST, Systems Research Laboratory, Dow Chemical , USA). One week after randomization, rats and mice were individually identified with a metal ear tag. Three days prior to initiating exposures both rats and mice were weighed and statistical analyses conducted to insure homogeneity of variance of group body weights and equality of group mean body weight. Statistical differences in group mean body weights were corrected (necessary only with male rats) by replacing the appropriate group with an extra group assigned identical ear tag numbers. The randomization, ear tagging and initial weighing were performed during a 12-14 day acclimation period. All animals were initially housed 2 or 3 per cage to facilitate adaption to the automatic watering system; they were housed singly after being ear tagged. A standard laboratory diet (Purina Laboratory Chow, Ralston Purina Co., St. Louis, MO) and water were offered -ad 1i bi tum except during exposure. Animals in the exposure groups were housed in separate, identical rooms with their respective inhalation chambers. Control animals were housed in a separate, similar room with no inhalation chamber.

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
Chambers
Exposures were conducted in stainless steel and glass inhalation chambers (4.3 m 3 ) with pyramidal-shaped ceilings. Temperature and relative humidity in the chambers were controlled by a system designed to maintain temperature at approximately 70°F and relative humidity at approximately 50%. Temperature and relative humidity in the chambers were recorded daily while exposures were in progress. All chambers were operated under dynamic airflow conditions and maintained at a slight negative pressure relative to the surrounding area. Control animals were not placed in an inhalation chamber due to the shortage of available chambers.

Vapor Generation and Analysis
Animals were exposed to 0, 400, 800 or 1600 ppm (0, 1.18, 2.35 or 4.71 mg.liter -1 ) butylene oxide vapors 6 hours per day for a total of 9 days
during an 11 day interval ; the animals were necropsied on the 12th day of the study. Exposure levels o f butylene oxide were generated by metering the liquid test substance at calculated rates into glass vaporization tubes. Vapors from the tubes were swept nto the chamber inlet ducts with compressed air. The compressed a i r was preheated with a flameless heat torch (Master, FHT-4) to facilitate complete vaporization. Total chamber airflow was maintained at approximately 800 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 i n each chamber was measured 2-3 times each hour by infrared spectroscopy using a Miran* I infrared gas analyzer at a wavelength of 11 u. The concentration of butylene oxide in the chamber was interpolated from standard curves derived from standards (100 liter SARAN** bags) of known concentration. The standardization was checked daily prior toinitiating exposures. A time-weighted average (TWA) analytical concentration was computed daily for each exposure leve1 .
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
5 days/week, for a total of 9 days over an 11 day period
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 400, 800 or 1600 ppm (0, 1.18, 2.35 or 4.71 mg liter-1 )
Basis:
other: targeted concentrations
Remarks:
Doses / Concentrations:
0, 400 +/- 5, 798 +/-17 or 1600 +/- 14 ppm
Basis:
other: time-weighted average (TWA) analytical concentrations
No. of animals per sex per dose:
5/sex/species/dose
Control animals:
yes, concurrent no treatment
Details on study design:
Male and female Fischer 344 rats and B6C3F1 mice to 0, 400, 800 or 1600 pprn butylene oxide vapors 6 hours per day, 5 days per week, for a total of 9 days during an 11 day interval. 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. The results of this study were used to design a 90-day 1,2-butylene oxide vapor inhalation study.

Examinations

Observations and examinations performed and frequency:
Animal Observations
All animals were observed daily for signs of toxicity and changes in appearance or demeanor. Body weights were recorded for each animal immediately prior to the first exposure, and again immediately prior to the 3rd, 5th and 7th days of exposure. Final body weights were recorded
immediately prior to sacrifice approximately 18 hours after the final exposure.
Sacrifice and pathology:
Hematology-Packed cell volume (PCV) , hemoglobin (Hgb) , red blood cell counts ( RBC ) , white blood cell counts (WBC) and differential white
cell counts were evaluated for all surviving rats and mice. Rat blood samples were taken by tail venipuncture immediately prior to the 8th
exposure day. Mouse blood samples were taken by orbital sinus puncture immediately prior to sacrifice.

Clinical Chemistry-Serum glucose ( Glu) , serum urea nitrogen (BUN), serum alkaline phosphatase activity (AP) and serum glutamic pyruvi c
transaminase activity (SGPT) were measured for allsurviving rats and mice. Rat blood samples were taken from severed cervical blood vessel s a t the
time of sacrifice; mouse blood samples were taken by orbital sinus puncture immediately prior to sacrifice.

Urinalysis- Specific gravity, pH, sugar, protein, ketones, blood, bilirubin and urobilinogen were measured on urine from male and female rats. Urine samples were taken from the rats immediately prior to the 8th day of exposure at the same time blood samples were taken for hematology.
Addi tionaf urine samples were taken from female rats immediately prior to sacrifice in order to recheck observed differences.

Gross Necropsy- Each animal was examined external ly and internal ly by a veterinary pathologist. Rats ( but not mice) were deprived of food overnight prior to sacrifice. Each animal was decapitated after clamping the trachea under methoxyflurane anesthesia; mice were exsaugui nated via the orbi tal sinus prior to decapitation. Lungs and trachea were removed as a unit and filled to approximately their normal inspiratory volume w i t h buffered 10% formalin. The nasal cavity was flushed with buffered 10% formalin to improve fixation of nasal turbinates prior to placing the entire head in formalin fixative. Weights of heart, liver, kidneys, brain, thymus, and testes (males) were recorded for each animal. The eyes of all animals were examined by a glass slide technique with fluorescent illumination. The head and vertebral bone were decalcified. Representative portions of an extensive list oforgans and tissues were taken from each animal and preserved in buffered 10% formalin.

Histopathology- The formal i n - fixed ti ssues were prepared by conventional histologic techniques, stained with hematoxyl in and eosin, and examined by light microscopy. A complete set of tissues from r a t s and mice in the control and high exposure groups were prepared for histopathologic examination. Only selected tissues, based on observations in the high exposure group, were examined for animals in the lower exposure groups i n order to ascertain whether or not there was a dose-response relationship. There were some instances when a few minor tissues were not present on slides for examination; these were not considered essential to the overall interpretation of the study and therefore additional sections were not
prepared.
Statistics:
Body weight data, organ weights, organ-to-body weight ratios,urinalysis values, hematological values and clinical chemistry values were evaluated by
analysis o f variance; differences between treatment group means and control group means were delineated by Dunnett's test (Steel and Torrie, 1960).Variances of group body weights were analyzed by Bartlett ' s test (Snedecor and Cochran, 1967). The level of significance chosen i n all cases was p <0.05.

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 examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
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:
no effects observed
Details on results:
Chamber Atmosphere Analysis
The mean TWA and mean nominal concentrations were within a very close range of the target concentration for each chamber; the close agreement between the mean TWA and mean nominal concentrations indicates that material losses were minimal in the vapor generating and exposure systems. The ranges of the mean daily TWA and nominal values and the coefficient of variation of the daily TWA measurements indicate that the test atmospheres were very stable and consistent on a day-to-day basis.
The mean temperature was approximately 70°F and the mean relataive humidity was approximately 50% for each exposure group.

Animal Observations
All 5 female mice and 3 of 5 male mice died during the 1 st day of exposure to 1600 ppm. Respiration was labored and was characterized by gasping through the mouth for one of the surviving male mice in the 1600 ppm group; this animal was found dead prior to the 2nd day of exposure, and the other was dead prior to the 3rd day of exposure. None of the rats died spontaneously during the course of the study. Rats in the 1600 pprn exposure group were noticeably smaller than controls by the time exposures were terminated.

Body Weights
Body weights of male and female rats in the 1600 ppm exposure group were markedly lower than for controls. In fact , the male rats in the 1600 ppm group gained only 0.2 g on the average during the exposure interval (control males gained 34.4 g); the 1600 pprn female rats lost 10.9 g on the average while control females gained 17.5 g. The body weights of 1600 ppm rats became statistically lower than for controls after 2 days of exposure and remained lower than controls thereafter. Mean body weights of male rats in the 800 ppm groups were also statistically lower than for controls, although the reduction in weight gain was not as pronounced as for animals in the high exposure group. There was also a trend toward reduced body weights for male rats in the 400 ppm group, with the mean body weights of the 400 pprn males being statistically different from controls after 2 days of exposure as well as at termination (fasted body weights). The body weights of female rats in the 400 and 800 ppm groups did not differ statistically from controls during the course o f the study or at termination. Although the absolute weights of male and female mice i n the 800 ppm exposure group were not statistically differentt from controls, both male and female mice i n the 800 ppm group as well as female mice in the 400 ppm group lost weight on the average during the course of the study while male and female control mice and 400 ppm male mice gained slightly.
.
Organ Weights
There were a variety o f absolute or relative organ weight changes for rats in the 1600 ppm group which were statistically significant, and some similar but less pronounced organ weight changes for rats in the 800 ppm group. These organ weight changes were, in general, considered to be a reflection of reduced body weight gain or stress rather than direct effects of the test material. Organ weights for mice in the 1600 ppm group were not measured since these animals died early in the course of the study. The mean absolute and relative kidney weights, mean heart weights and mean absolute and relative thymus weights of male mice i n the 800 ppm group, as well as the mean absolute and relative thymus weights of female mice in the
800 ppm group, were statistically different from controls; these results were considered to be reflections of the weight loss or stress rather than direct effects of the test material. The statistical differences for absolute and relative liver weights between control and 400 ppm female mice were considered to be a sporadic occurrence unrelated to exposure in view of the absence of effects on liver weights forr female mice in the higher (800 ppm) exposure group.

Hematology
Mean white blood cell counts of male and female rats in the 1600 ppm exposure group were higher than for controls. Differential white cell counts for 1600 ppm male and female rats indicated a trend toward a decreased percentage of lymphocytes with an increased percentage of neutrophils. In addition, the mean hemoglobin value was statistically higher for the 1600 ppm female rats than for controls, but was considered withinj the range of normal biological variability and not of toxicological significance since red blood cell counts and packed cell volume were unaltered. No hematologic analyses were performed for 1600 ppm mice since mice in that group died prior to the time hematology evaluations were performed. The mean packed cell volume of the 800 ppm female mice was statistically lower than for controls. However, the significance of this observation is uncertain in view of the fact that red cell counts and hemoglobin values of the same animals were within the normal range. Moreover, the white blood cell counts were statistically higher than controls for female mice in both the 400 and 800 ppm groups. However, the mean white blood cell count for female control mice was low compared to historical control data, while the mean values for treated groups of mice were very similar to historical control values. Differential white counts for 800 ppm female mice indicated the same trend toward an increased percentage of neutrophils and a decreased percentage of lymphocytes as observed in 1600 ppm rats; no such trend was apparent for male mice. The mean white cell count and mean hemoglobin values for male mice in the 400 ppm group differed statistically from control values, but these were apparently sporadic results unrelated to exposure since there were no affects on these parameters in 800 ppm male mice.

Clinical Chemistry
Serum alkaline phosphatase (AP) activity was depressed in both male and female rats in the 800 and 1600 ppm groups. Although such depressions of serum AP activity have no known toxicologic significance, they could be a reflection of decreased AP activity of intestinal origin associated with decreased food intake or a generalized nutritional protein deficiency as further indicated by the retarded growth of these animals. There were no other apparent effects on clinical chemistry parameters of rats. Alkaline phosphatase activity was also depressed for male mice in the 800 ppm groups as well as for females i n the 400 and 800 ppm groups. As for rats , these depressions of alkaline phosphatase activity, along with the lower mean glucose values for male and female mice in the 800 ppm group, are probably indirectly related to.exposure, reflecting a generalized impairment of nutritional status and growth. Blood urea nitrogen (BUN) values of female mice in the 800 ppm group were significantly lower than for controls, possibly also as a result of a generalized nutritional deficiency. However, decreases in BUN values do not reflect renal toxicity, but may be indicative of generalized nutritional status.

Urinalysis
There were no apparent treatment-related alterations of urinary parameters for male rats. Specific gravity of urine from female rats in the 800 and 1600 ppm groups was found to be significantly higher than for controls. Analysis of additional urine samples from fasted female rats taken immediately prior to sacrific verified that there was an exposure-related increase in specific gravity for the 800 and 1600 ppm females. The first analysis also indicated a very slight trend toward increased protein in urine from females, but this was not verified in the repeat examination. There were no apparent alterations of other urinalysis parameters (e.g., glucose, pH, protein, etc.) or any other indications of kidney toxicity in either rats or mice.

Gross P athology
-Rats. There was a tendency toward a greater incidence of focal corneal cloudiness in the 800 and 1600 ppm exposure groups, possibly as a result of exposure to the test material. One male rat in the high exposure group had a focal gastric ulcer with edema. All other observations in rats, including decreased size of abdominal organs, abdominal fat , thoracic viscera, thymus, mediastinal fat or mesenteric tissue in a t least some animals in each exposure group, were considered to be reflections of decreased growth rate or stress rather than direct effects of the test material.

-Mice. The gross pathologic observations i n mice were similar to those in rats. No specific cause of death could be identified upon gross pathologic examinations of mice in the 1600 ppm group. Occasional, small, darkened foci were found in the lungs of one male mouse in the 1600 ppm group. Alll other gross pathologic observations in male and female mice, including decreased amount of abdominal fat, decreased size of abdominal organs, thymus or mediastinal tissue in at least some mice in each exposure group, were considered to be reflections of decreased growth rate or stress rather than direct effects of the test material.

Histopathology
-Rats. Treatment-related microscopic changes in the nasal turbinates, including inflamatory and degenerative changes, were noted for male and female rats in the 800 and 1600 ppm exposure groups. Both the olfactory and respiratory portions of the nasal mucosa were affected. Although there was no evidence of treatment-related effects in the lungs of these animals, shortening of the tracheal epithelium with a polymorphonuclear inflammatory cell infiltrate was noted in one male rat in the 1600 ppm exposure group. Myeloid hyperplasia was observed in vertebral bone marrow of most 1600 ppm animals and a few 800 ppm animals, probably as a result of inflammation in the nasal mucosa. All other microscopic observations in rats were considered to be either spontaneous in nature and unrelated to exposure, or reflections of growth retardation or stress.

-Mice. Since all of the mice in the 1600 ppm group died prior to completion of the exposures, complete sets of tissues were examined microscopically for 800 ppm animals rather than 1600 ppm animals; only selected tissues from 400 and 1600 ppm exposure groups were examined. As in rats, treatment-related changes in the nasal turbinates were detected in male and female mice in the 800 and 1600 ppm exposure groups. Flattening of the olfactory epithelium was noted in all female mice and 2 of 5 male mice in the 800 ppm group. No epithelial changes in respiratory epithelium of mice were apparent, but exudative rhinitis (inflammatory cells migrating through the epithelial layer) was present throughout the nasal turbinate lumen in many of the mice i n 800 and 1600 ppm groups, as well as in 1 of 5 female mice in the 400 ppm group. Myeloid hyperplasia was observed in vertebral
bone marrow of all 800 ppm mice, probably as a result of inflammation in the nasal mucosa. All other microscopic observations were considered to be either spontaneous in nature and unrelated to exposure, or indirectly related to exposure via decreased food consumption or stress.

Effect levels

Basis for effect level:
other: Based on the study results, exposure levels of 0, 75, 150 and 600 ppm were selected for a subchronic vapor inhalation study.
Remarks on result:
not measured/tested
Remarks:
Effect level not specified (migrated information)

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the study results, exposure levels of 0, 75, 150 and 600 ppm were selected for a subchronic vapor inhalation study.