1,2-epoxybutane

Endpoint summary

Description of key information

Key (similar to OECD TG 413 in rats and mice): NOAEC for both species and both genders = 150 ppm

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

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

Reference

Reference 1

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

GLP compliance:

yes

Limit test:

no

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

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).

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

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.

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.

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

Key result

Critical effects observed:

no

Reference 2

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

B6C3F1

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, 2 or 3 per cage for several days in order to facilitate drinking behavior from automatic watering devices, and then they were housed singly.
- 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

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).

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 and mice were exposed via inhalation to 0, 75, 150 or 600 ppm 1,2-butylene oxide vapors (0.22, 0.44 or 1.77 mg liter-1) 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

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
Mouse blood samples were taken by orbital sinus puncture immediately prior to 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
Mouse blood samples were taken from nonfasted animals by orbital sinus puncture at time of sacrifice. 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: No

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. Mice were not deprived of food overnight prior to necropsy. Mice were anesthetized with methoxyflurane and exsanguinated via the orbital sinus and then killed by decapitation. 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.

Clinical signs:

no effects observed

Description (incidence and severity):

One male mouse in the 150 ppm group died spontaneously during the course of the study. Gross pathologic observations for this animal indicated that the animal probably died as the result of a urethral obstruction unrelated to exposure. Otherwise, all mice appeared normal and healthy throughout the study.

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Growth of female mice 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 mice 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 male mice in the 75 ppm group was statistically lower than controls; this was considered to be a sporadic result unrelated to exposure in view of the absence of an observable effect on red cell counts for male mice in the two higher exposure groups. There were no other statistical differences for hematologic parameters of mice after 30 days of exposure.
90-Day Sacrifice: For male mice, the mean white blood cell count for animals in the 75 and 600 ppm groups were statistically higher than for controls. However, the mean white blood cell counts of treatment groups of male mice were within the range of historical control group values and there was no dose-response relationship. Therefore, the statistical differences for white cell counts in male mice in the present study were considered to be sporadic occurrences unrelated to exposure to the test material. The white blood cell counts for treatment groups of female mice, on the other hand, were statistically lower than for controls. However, in this case, the mean white blood cell count for the female mice in the concurrent control group was higher than for historical control groups while the mean white cell counts for treatment groups were well within the range of historical control values. Therefore the statistical differences for white blood cell counts of female mice were considered to be unrelated to exposure, and of no toxicological significance.
The mean packed cell volume, mean red blood cell count, and mean hemoglobin concentration of female mice in the 150 and 600 ppm groups were also statistically lower than for controls. However, none of the hematologic parameters were dramatically altered, with only the mean packed cell volume and mean hemoglobin values of 600 ppm female mice being slightly lower than the range of historical control values. In addition, there was no clear dose-response relationship, since the mean values in the two treatment groups were very similar. Moreover, there was no microscopic evidence of bone marrow toxicity in mice. Hence, the statistical difference for hematologic parameters of female mice are probably of minimal, if any, toxicologic significance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

30-Day Interim Sacrifice: There were no statistical differences or apparent effects on clinical chemistry parameters of male or female mice after 30 days of exposure.
90-Day Sacrifice: There were no statistical differences or apparent effects on clinical chemistry parameters of male or female mice. Historical control values were obtained from control group animals in five recently conducted inhalation studies of similar design.

Endocrine findings:

not examined

Urinalysis findings:

not examined

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 mice 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 mice were sporadic in nature and lacked a dose-response relationship, or were reflections of depressed body weight gain.
90-Day Sacrifice: The various statistically significant organ weight changes for male and female mice in the 600 ppm group were considered to be reflections of reduced body weight gain rather than direct toxic effects of exposure to butylene oxide vapors.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

30-Day Interim Sacrifice. There were no gross pathologic observations in male or female mice after 30 days of exposure which were considered to be related to exposure.
90-Day Sacrifice. Most male and female mice in the 600 ppm group had a decreased amount of fat within the abdominal cavity. Decreased amounts of fat in the mediastinum as well as decreased size of the thymus gland was noted in some 600 ppm mice after 90 days of exposure. All other gross pathologic observations in mice were considered to be spontaneous in nature and unrelated to exposure. No treatment related changes were detected in mice in the 75 and 150 pp groups after 90 days of exposure.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathologic examinations of tissues from mice after 90 days of exposure revealed changes only in the nasal mucosa of 600 ppm animals which were considered to be direct effects of exposure to the test material. The microscopic changes in the nasal turbinates were characterized by a minimal degree of focal thickening and flattening of the respiratory epithelium. There were increased numbers of inflammatory cells present in the nasal mucosa and within the lumen of the nasal cavity; no treatment-related changes were noted in lungs or trachea of mice. There were several other microscopic changes in 600 ppm mice which were considered to be indirect effects of exposure to the test material including decreased hepatocellular size and decreased cell content in the cortex of the thymus gland. All other microscopic observations were considered to be spontaneous in nature and unrelated to exposure. There were no microscopic observations in either male or female mice in the 75 and 150 ppm exposure groups which were considered to be related to exposure to the test material. Since only minor treatment-related effects were detected microscopically after 90 days of exposure, microscopic examinations were not performed on tissues from mice sacrificed after 30 days of exposure.

Histopathological findings: neoplastic:

no effects observed

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.

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

Key result

Critical effects observed:

no

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

150 ppm

Study duration:

subchronic

Species:

rat

Quality of whole database:

similar to OECD TG 413

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Reference 1

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

GLP compliance:

yes

Limit test:

no

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

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).

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

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.

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.

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

Key result

Critical effects observed:

no

Reference 2

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

B6C3F1

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, 2 or 3 per cage for several days in order to facilitate drinking behavior from automatic watering devices, and then they were housed singly.
- 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

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).

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 and mice were exposed via inhalation to 0, 75, 150 or 600 ppm 1,2-butylene oxide vapors (0.22, 0.44 or 1.77 mg liter-1) 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

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
Mouse blood samples were taken by orbital sinus puncture immediately prior to 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
Mouse blood samples were taken from nonfasted animals by orbital sinus puncture at time of sacrifice. 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: No

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. Mice were not deprived of food overnight prior to necropsy. Mice were anesthetized with methoxyflurane and exsanguinated via the orbital sinus and then killed by decapitation. 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.

Clinical signs:

no effects observed

Description (incidence and severity):

One male mouse in the 150 ppm group died spontaneously during the course of the study. Gross pathologic observations for this animal indicated that the animal probably died as the result of a urethral obstruction unrelated to exposure. Otherwise, all mice appeared normal and healthy throughout the study.

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Growth of female mice 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 mice 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 male mice in the 75 ppm group was statistically lower than controls; this was considered to be a sporadic result unrelated to exposure in view of the absence of an observable effect on red cell counts for male mice in the two higher exposure groups. There were no other statistical differences for hematologic parameters of mice after 30 days of exposure.
90-Day Sacrifice: For male mice, the mean white blood cell count for animals in the 75 and 600 ppm groups were statistically higher than for controls. However, the mean white blood cell counts of treatment groups of male mice were within the range of historical control group values and there was no dose-response relationship. Therefore, the statistical differences for white cell counts in male mice in the present study were considered to be sporadic occurrences unrelated to exposure to the test material. The white blood cell counts for treatment groups of female mice, on the other hand, were statistically lower than for controls. However, in this case, the mean white blood cell count for the female mice in the concurrent control group was higher than for historical control groups while the mean white cell counts for treatment groups were well within the range of historical control values. Therefore the statistical differences for white blood cell counts of female mice were considered to be unrelated to exposure, and of no toxicological significance.
The mean packed cell volume, mean red blood cell count, and mean hemoglobin concentration of female mice in the 150 and 600 ppm groups were also statistically lower than for controls. However, none of the hematologic parameters were dramatically altered, with only the mean packed cell volume and mean hemoglobin values of 600 ppm female mice being slightly lower than the range of historical control values. In addition, there was no clear dose-response relationship, since the mean values in the two treatment groups were very similar. Moreover, there was no microscopic evidence of bone marrow toxicity in mice. Hence, the statistical difference for hematologic parameters of female mice are probably of minimal, if any, toxicologic significance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

30-Day Interim Sacrifice: There were no statistical differences or apparent effects on clinical chemistry parameters of male or female mice after 30 days of exposure.
90-Day Sacrifice: There were no statistical differences or apparent effects on clinical chemistry parameters of male or female mice. Historical control values were obtained from control group animals in five recently conducted inhalation studies of similar design.

Endocrine findings:

not examined

Urinalysis findings:

not examined

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 mice 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 mice were sporadic in nature and lacked a dose-response relationship, or were reflections of depressed body weight gain.
90-Day Sacrifice: The various statistically significant organ weight changes for male and female mice in the 600 ppm group were considered to be reflections of reduced body weight gain rather than direct toxic effects of exposure to butylene oxide vapors.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

30-Day Interim Sacrifice. There were no gross pathologic observations in male or female mice after 30 days of exposure which were considered to be related to exposure.
90-Day Sacrifice. Most male and female mice in the 600 ppm group had a decreased amount of fat within the abdominal cavity. Decreased amounts of fat in the mediastinum as well as decreased size of the thymus gland was noted in some 600 ppm mice after 90 days of exposure. All other gross pathologic observations in mice were considered to be spontaneous in nature and unrelated to exposure. No treatment related changes were detected in mice in the 75 and 150 pp groups after 90 days of exposure.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathologic examinations of tissues from mice after 90 days of exposure revealed changes only in the nasal mucosa of 600 ppm animals which were considered to be direct effects of exposure to the test material. The microscopic changes in the nasal turbinates were characterized by a minimal degree of focal thickening and flattening of the respiratory epithelium. There were increased numbers of inflammatory cells present in the nasal mucosa and within the lumen of the nasal cavity; no treatment-related changes were noted in lungs or trachea of mice. There were several other microscopic changes in 600 ppm mice which were considered to be indirect effects of exposure to the test material including decreased hepatocellular size and decreased cell content in the cortex of the thymus gland. All other microscopic observations were considered to be spontaneous in nature and unrelated to exposure. There were no microscopic observations in either male or female mice in the 75 and 150 ppm exposure groups which were considered to be related to exposure to the test material. Since only minor treatment-related effects were detected microscopically after 90 days of exposure, microscopic examinations were not performed on tissues from mice sacrificed after 30 days of exposure.

Histopathological findings: neoplastic:

no effects observed

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.

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

Key result

Critical effects observed:

no

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

150 ppm

Study duration:

subchronic

Species:

rat

Quality of whole database:

similar to OECD TG 413

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

A 90-day inhalation (whole body) study similar to OECD TG 413 is available for rats and mice (Dow, 1980). Fischer 344 rats and B6C3F1 mice (15/group) were exposed via inhalation to 0, 75, 150 or 600 ppm 1,2-butylene oxide 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.The tested concentrations based on results observed in a dose-range finding study, in which animals have been exposed to vapor concentrations of 400, 800 and 1600 ppm (Dow, 1980). All animals were observed daily for signs of toxicity and changes in appearance or behavior. Body weights were recorded. Hematology, clinical chemistry, urinalysis, gross necropsy and histopathology was conducted. The findings for of this study for rats and mice have been reported in Miller et al.1981.The mean body weight gain for female rats in the 600 ppm group was significantly lower than for controls during the last few weeks of study. In addition, the body weight gains of male rats in the 600 ppm group tended to be lower than for controls, although not statistically significantly. Growth of rats in the 75 and 150 ppm groups was not altered by the exposures. There were several statistically significant changes in absolute or relative organ weights of rats (particularly those in the 600 ppm group) after 30 days or 90 days of exposure which were considered to be reflections of reduced body weight gain or generalized stress, rather than direct toxic effects of the test material. Hematologic analyses for rats sacrificed after 4 weeks revealed no changes of toxicologic significance. After 13 weeks, there were no statistically significant differences and no apparent effects on the hematologic parameters of male rats. The mean hemoglobin values of female rats in the 150 ppm group, and the mean red blood cell count of female rats in the 150 ppm group were statistically significantly higher than for controls; these values were within the range of normal for female rats of the same strain and age, and were considered to be sporadic occurrences with no toxicologic significance. Clinical chemistry analyses for rats were unremarkable. Urinary parameters of rats were unaltered in rats sacrificed after 4 weeks. The mean urinary specific gravity values of male and female rats in the 600 ppm group were statistically significantly lower than for controls after 13 weeks. However, there were no apparent effects on any other urinary parameters measured, nor any other indications of nephrotoxicity in rats. Gross pathologic observations in rats in the 600 ppm group included decreased amounts of abdominal adipose tissue, and decreased size of the thymus and mediastinal fat. AII other gross pathologic observations in rats were considered spontaneous in nature and unrelated to exposure. There were no treatment-related gross pathologic observations in rats in the 75 or 150 ppm groups. Histopathologic examinations of tissues from rats after 13 weeks of exposure to 600 ppm revealed changes in the nasal mucosa which were attributed to primary upper respiratory irritation. The microscopic changes in the nasal turbinates were minimal and were characterized by flattening of the olfactory and respiratory epithelium with some focal thickening 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 (e.g., trachea and lungs) 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 rats exposed to 600 ppm 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 (3 of 10 male rats only). All other microscopic observations in rats 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 of urine in rats exposed to 600 ppm. 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. Since only minor pathologic treatment-related effects were detected microscopically after 13 weeks of exposure, microscopic examinations were not performed on tissues from rats sacrificed after 4-weeks of exposure. NOAEC for male and female rats is 150 ppm.

For mice, the following findings have been reported in Miller et al.1981: One male mouse in the 150 ppm group died spontaneously during the course of the study. Gross pathologic examination of this animal indicated that it probably died as a result of a urethral obstruction unrelated to exposure. All other mice survived and appeared normal and healthy throughout the study. The mean body weight gain for female mice in the 600 ppm group was significantly lower than for controls during the last few weeks of study. In addition, the body weight gains of male mice in the 600 ppm group tended to be lower than for controls, although not statistically significantly. Growth of mice in the 75 and 150 ppm groups was not altered by the exposures. The mean body weight gain values of male mice in the 75 ppm group were statistically lower than controls on several occasions, but this was not considered to be an adverse treatment-related effect due to the absence of similar changes for male mice in the 150 ppm group. There were several statistically significant changes in absolute or relative organ weights of mice (particularly those in the 600 ppm group) after 30 days or 90 days of exposure which were considered to be reflections of reduced body weight gain or generalized stress, rather than direct toxic effects of the test material. Hematologic analyses for mice sacrificed after 4 weeks revealed no changes of toxicologic significance. There were a variety of statistically significant differences for hematologic parameters of mice after 13 weeks. For males, the mean white blood cell counts of animals in the 150 and 600 ppm groups were statistically higher than for controls. However, the mean white blood cell counts of treatment groups of male mice were within the range of normal values for animals of the same strain and age, and there was no dose-response relationship. Therefore, the statistical differences for white blood cell counts in male mice were thought to be reflections of normal biological variability. The white blood cell counts for treatment groups of female mice, on the other hand, were statistically lower than for controls. However, in this case, the mean white blood cell count for the female mice in the concurrent control group was higher than for historical control groups while the mean white cell counts for treatment groups were well within the range of historical control values. Therefore the statistical differences for white blood cell counts of female mice were considered to unrelated to exposure, and of no toxicological significance. The mean packed cell volume, mean red blood cell count, and mean hemoglobin concentration of female mice in the 150 and 600 ppm groups were also statistically lower than for controls. However, none of the hematologic parameters were analytically altered, with only the mean packed cell volume and mean hemoglobin values of 600 ppm female mice being slightly lower than the range of historical control values. In addition, there was no clear dose-response relationship, since the mean values in the two treatment groups were very similar. Moreover, there was no microscopic evidence of bone marrow toxicity in mice. Hence, the statistical differences for hematologic parameters of male mice are probably of minimal, if any, toxicologic significance. Clinical chemistry analyses for mice were unremarkable. However, there were no apparent effects on any other urinary parameters measured, nor any other indications of nephrotoxicity in mice. Gross pathologic observations in mice in the 600 ppm group included decreased amounts of abdominal adipose tissue, and decreased size of the thymus and mediastinal fat. All other gross pathologic observations in mice were considered spontaneous in nature and unrelated to exposure. There were no treatment-related gross pathologic observations in mice in the 75 or 150 ppm groups. Histopathologic examinations of tissues from mice after 13-weeks of exposure also revealed changes only in the nasal mucosa of animals exposed to 600 ppm which were considered to be direct effects of exposure to the test material. The microscopic changes in the nasal turbinates were characterized by a minimal degree of focal thickening and flattening of the respiratory epithelium. There were increased numbers of inflammatory cells present in the nasal mucosa and within the lumen of the nasal cavity; no treatment-related changes were noted in the lungs or trachea of mice. Also, there were several other microscopic changes in mice exposed to 600 ppm which were considered to be indirect effects of exposure to the test material including decreased hepatocellular size and decreased cell content in the cortex of the thymus gland. All other microscopic observations in mice were considered to be spontaneous in nature and unrelated to exposure. There were no microscopic observations in either male or female mice in the 75 and 150 ppm exposure groups which were considered to be related to exposure to the test material. Since only minor treatment-related effects were detected microscopically after 1 3-weeks of exposure, microscopic examinations were not performed on tissues from mice sacrificed after 4 weeks of exposure. NOAEC for male and female mice is 150 ppm.

In a further supporting study (NTP,1988), groups of 10 male and female rats were exposed to 50, 100, 200, 400 and 800 ppm test item vapor via inhalation. Exposure was conducted for 6 h/d, 5 d/w (65 exposures) over a study period of 13 weeks. Animals were observed twice daily for clinical signs and mortality. Body weight was recorded on the first day of exposure, once weekly thereafter and at necropsy. Necropsy was performed on all animals. All control and high dose groups were examined histopathologically. No compound-related deaths occurred. The final mean body weight of rats exposed at 800 ppm was 23% lower than that of the controls for males and 16% lower for females. No compound-related clinical signs were observed. Liver weight to body weight ratios were similar in dosed and control rats. Inflammation of the nasal cavity was seen in all rats that received 1,2-epoxybutane at 800 ppm but not at lower concentrations. The inflammation was present primarily in the dorsal and lateral portions of the nasal cavity and affected the respiratory and olfactory epithelium. The lesion was characterized by lymphocytic and neutrophilic infiltration of the mucosa and accumulation of purulent exudate in the lumen of the nasal cavity, with focal loss of epithelial cells from the mucosa. The NOAEC for males and females was 400 ppm.

A NTP study is also available for male and female mice (NTP, 1988). Groups of 10 male and female rats were exposed to 50, 100, 200, 400 and 800 ppm test item vapor via inhalation. Exposure was conducted for 6 h/d, 5 d/w (65 exposures) over a study period of 13 weeks. Body weight was recorded on the first day of exposure, once weekly thereafter and at necropsy. Necropsy was performed on all animals. All control and high dose groups were examined histopathologically. All mice exposed at 800 ppm died before the end of the studies. Final mean body weights were not affected by exposure to 1,2-epoxybutane. Mice exposed at 800 ppm were listless during and after the first day of exposure. Clinical signs were not seen at lower doses. The liver weight to body weight ratio of female mice that received 400 ppm was significantly lower than that of the controls. Renal tubular necrosis was seen in 6/10 males and 8/10 females exposed at 800 ppm but not at lower exposure concentrations. Inflammation of the nasal turbinates was observed in all mice exposed at 200 ppm or higher, in 0/10 males and 7/10 females exposed at 100 ppm, and in none of the controls. Renal and upper respiratory tract changes were considered to be compound related. The NOAEC for females is 50 ppm based on upper respiratory tract lesions and the NOAEC for males is 100 ppm.

Three supporting chronic inhalation studies are available for rats, mice and guinea pigs. Four groups of animals (10/group/sex rats, 8/group/sex guinea pigs and 2/group/sex rabbits) were exposed to vapors of the test item (whole body). One group received 135 to 141 seven-hour exposures over a period of 198 days to an average concentration of 800 ppm (2.34 mg/L) of butylene oxide(s). The second group was exposed in the same manner to 400 ppm (1.18 mg/L) of butylene oxide(s). The third and fourth groups were held at all times in the animal quarters and served as the unexposed controls (Dow, 1961). During the course of the exposures, the animals were observed frequently as to general appearance, health and behavior. Moribund animals were sacrificed in an attempt to ascertain the cause of illness. Body weight was recorded. Near the end of the exposures, hematological studies were made upon selected rats and guinea pigs of both sexes. These studies consisted of total red and white cell counts, hemoglobin content and differential white cell counts. Blood-urea-nitrogen determinations were made on selected rats and rabbits at both levels and on controls. In addition, urinalyses were conducted on selected control rats of both sexes and on selected animals receiving exposures to the 800 ppm level only. The urinalyses were made on the urine pooled from groups of rats. At the termination of the exposures all surviving animals were sacrificed and the tissues examined grossly. Sections of preserved organs were made and stained with hematoxylin-eosin for histopathological examination. There were evidences of adverse effects to the rats, rabbits and guinea pigs that received exposures to an average concentration of 800 ppm. These effects were: depression of growth of the male rats, a marked increase in mortality in the male rats which was ascribed to lung infections resulting from irritation due to the exposure and a significant increase in average lung weights in the rats of both sexes. In guinea pigs which have been exposed to 800 ppm, depression of growth of males and significant depression of average final body weight of the males was reported. There was a significant increase in average lung weights in the guinea pigs of both sexes at the highest dose. In addition, there was a significant decrease in the average weight of the livers of the female guinea pigs. No evidence of adverse effects to rats, rabbits and guinea pigs of both sexes was reported for the 400 ppm group as judged by general appearance and behavior, mortality, gross and microscopic examination of tissues, final average body and organ weights  blood-urea-nitrogen values or hematological values from representative rats. Blood-urea-nitrogen values were within normal limits of variation for the rats and rabbits of both sexes. Albumin was demonstrated in the urine from the exposed male rats only. The significance of this finding is not known as there were no abnormal gross or histopathological findings in any of the organs, including the kidneys, in any of the experimental animals. These results indicate that the test item of the composition studied have a moderate degree of toxicity from repeated vapor exposures.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. After repeated inhalation local irritation was observed as main effect. Since the test substance is harmonized classified according for STOT SE 3 (H334), no further classification for repeated dose toxicity is warranted.