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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
repeated dose toxicity: inhalation
Remarks:
other: reproduction
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Name of test material (as cited in study report): Isobutanol
- Physical state: liquid
- Analytical purity: 99.9 %
- Stability under test conditions: the test substance was proven stable under the storage conditions
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc.
- Age at study initiation: (P) ca. 7 wks; (F1) ca. 4 wks
- Weight at study initiation: (P) Males: 236-350 g; Females: 159-213 g; (F1) Males: post natal day 32: means: 84-97 g; Females: post natal day 32: means: 78-88 g
- Housing: individually
- Diet: ad libitum (no food during exposure)
- Water: ad libitum (no water during exposure)
- Acclimation period: 21 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3 °C
- Humidity (%): 30-70 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Exposures were conducted in four 2.0 m3 stainless steel and glass whole-body inhalation chambers. One chamber was dedicated for each group for the duration of the study. Chamber supply air was provided from a HEPA- and charcoal-filtered, temperature- and humidity-controlled source. Treatment of exhaust air consisted of charcoal- and HEPA filtration.

The generation system was operated as follows: Vapors of the test article were generated using a heated bead column. The test article was introduced to the top of the column, while nitrogen entering the bottom of the column served as the carrier gas. For Groups 2 and 3 the column was 2.4 cm in diameter (ID), 40 cm long filled with 2, 3 and 4 mm beads. For Group 4, the column was 5 cm in diameter (ID) and 68 cm long filled with 3, 6, 8 and 12-mm beads. The columns were wrapped with heating tapes (Omega Engineering). Temperatures were set to approximately 100°C for chambers 2 and 3, and approximately 170-185ºC for chamber 4 using an Omega CN370 temperature controller. The chamber 4 temperature was significantly higher due to the need to maintain an internal temperature of at least 70ºC to ensure vaporization of the test article. The test article was vaporized as it dripped from 1/16-inch Teflon tubing onto the glass beads contained within each heated column.
The test article was metered from an amber glass reservoir to the column using an FMI pump (Fluid Metering, Inc., Oyster Bay, New York). Calibrated FMI pumps included 2 model no. QG-6 pumps with a 1/4-inch piston for chamber 2, and a 3/8-inch piston for chamber 3 and a model no. QG-20 pump with a 1/4-inch piston for chamber 4.
Vaporization nitrogen was delivered from the facility nitrogen generation system and was controlled using calibrated flowmeters (Gilmont Instruments, Barrington, Illinois). The vaporization nitrogen carried the isobutanol vapor through Teflon delivery lines (3/8-inch O.D. tubing for chambers 2 and 3, 1-inch O.D. tubing for chamber 4) to the chamber inlet where the concentration was reduced to the desired level with chamber ventilation air. The animal exposure was initiated by switching the FMI pumps and the compressed nitrogen on simultaneously.

TEST ATMOSPHERE
- Brief description of analytical method used: Exposure concentrations within each chamber were measured 9 to 10 times (approximately every 35 minutes) during each daily exposure period by a validated gas chromatographic method. At least one standard was analyzed each day prior to exposure to confirm gas chromatographic calibration. Chamber temperature, relative humidity, ventilation rate, and negative pressure within the chambers were monitored continuously and were recorded approximately every 35 minutes. Oxygen content within the chamber was measured during the pre-study method development phase. Nominal chamber concentrations were determined daily. Total air volume was calculated by multiplying mean chamber ventilation rate (in liters per minute) by the exposure duration (in minutes). Test atmosphere homogeneity was demonstrated during pre-study method development. There were no detectable aerosols at any evaluation interval.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Exposure concentrations within each chamber were measured 9 to 10 times (approximately every 35 minutes) during each daily exposure period by a validated gas chromatographic method.
Duration of treatment / exposure:
for the F0/P generation: approx. 17 weeks (beginning at least 70 days before mating until postweaning)
Frequency of treatment:
daily for 6 hours per day
Doses / concentrationsopen allclose all
Dose / conc.:
500 ppm
Dose / conc.:
1 000 ppm
Dose / conc.:
2 500 ppm
No. of animals per sex per dose:
30
Control animals:
yes

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS/DETAILED CLINICAL OBSERVATIONS
Detailed physical examinations were recorded weekly for all parental animals throughout the study period. All animals were observed twice daily for moribundity and mortality; in addition, the animals were observed for appearance, behavior and pharmacotoxic signs within one hour after completion of exposure. Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties.

BODY WEIGHT
Time schedule for examinations: Individual F0 and F1 male body weights were recorded on study days 0, 1, 4 and 7, weekly thereafter throughout the study, and prior to the scheduled necropsy. Individual F0 and F1 female body weights were recorded on study days 0, 1, 4 and 7 and weekly thereafter until evidence of copulation was observed. Mean weekly body weights and body weight changes are presented for each interval. Once evidence of mating was observed, female body weights were recorded on gestation days 0, 4, 7, 11, 14 and 20 and on lactation days 1, 4, 7, 14 and 21; for the F0 females, body weights were also recorded on lactation day 28. Body weight changes are presented for each of these intervals. After weaning (lactation day 28), weekly body weights were recorded for these F0 females until the scheduled necropsy.

FOOD CONSUMPTION
Individual F0 and F1 male and female food consumption was measured on study days 0, 1, 4 and 7 and weekly thereafter until pairing. Food intake was not recorded during the mating period. Male food consumption was measured after mating on a weekly basis until the scheduled necropsy. Female food consumption was recorded on gestation days 0, 4, 7, 11, 14 and 20 and lactation days 1, 4, 7, 14 and 21; for the F0 females, food consumption was also recorded on lactation day 28. For the F0 generation, the last scheduled interval for weekly recording of food consumption was study day 126. Since final body weights and clinical observations were recorded on the scheduled necropsy days (after study day 126), food consumption was manually recorded at that time. These data are not presented in the report tables, but will be maintained in the raw data. Food consumption was calculated and reported as g/animal/day and g/kg/day for the corresponding body weight change intervals. Food efficiency (body weight gained as a percentage of food consumed) was also calculated and reported for these intervals.
Sacrifice and pathology:
SACRIFICE
- Male animals: All surviving adults were euthanized following the selection of the F1 generation and completion of a detailed clinical observation.
- Maternal animals: All surviving adults were euthanized following the selection of the F1 generation and completion of a detailed clinical observation.

GROSS NECROPSY
The necropsy included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal and pelvic cavities including viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
organs collected: Adrenals (2); Aorta; Bone with marrow (sternebrae); Brain (forebrain, midbrain, hindbrain); Coagulating gland; Eyes with optic nerve (2); Gastrointestinal tract (Esophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum); Heart; Kidneys (2); Liver (sections of two lobes); Lungs (including bronchi, fixed by inflation with fixative); Lymph node (mesenteric); Ovaries and oviducts (2); Pancreas; Peripheral nerve (sciatic); Pituitary; Prostate; Salivary gland [submandibular (2)]; Seminal vesicles (2); Skeletal muscle (rectus femoris); Skin with mammary gland; Spinal cord (cervical); Spleen; Testes with epididymidesa (the right testis and epididymis were fixed in Bouin's solution) and vas deferens; Thymus; Thyroids [with parathyroids, if present (2)]; Trachea; Urinary bladder; Uterus with cervix and vagina; All gross lesions
Organ weights: Adrenals; Brain; Epididymides (total and caudal; these paired organs were weighed seperately); Kidneys; Liver; Ovaries; Pituitary; Prostate; Seminal vesicles with coagulating glands (with accessory fluids), Spleen, Testes (these paired organs were weighed seperately), Thymus gland; Uterus with oviducts and cervix
Organs examined: Adrenal glands: cortex and medulla; Brain; Cervix; Epididymis (right): caput, corpus and cauda; Kidneys; Liver; Ovaries; Pituitary, Prostate; Seminal vesivles with coagulatin glands (with accessory fluids); Spleen; Testis (right); Thymus, Uterus (with oviducts); Vagina; All gross (internal) lesions
Statistics:
see chapter Toxicity on Reproduction

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
PARENTAL ANIMALS
After 91 days of exposure to the test article, female no. 74805 in the 500 ppm group had pale eyes and ears and appeared to be having difficulty during parturition (gestation day 23) and was euthanized in extremis on lactation day 0. This female delivered 12 pups and had four pups retained in utero. On the day of euthanasia, this female had pale ears and eyes and red discharge from the vagina. Microscopically, the cause of death for this female was determined to be severe acute renal tubular necrosis and moderate acute hepatic necrosis. Therefore, this death was not attributed to the test article; no evidence of dystocia was observed in females in the 1000 or 2500 ppm groups. All other animals survived to the scheduled necropsy.
There were no test article-related clinical observations at the weekly detailed physical examinations or one hour following exposure. The response to novel stimulus was similar in all groups, including the control group.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
PARENTAL ANIMALS
Mean F0 body weights, body weight gains and cumulative body weight gains in the 500,
1000 and 2500 ppm groups were unaffected by exposure to the test article during the premating period and one week following weaning (females) and throughout the study (males). The only statistically significant (p<0.05) difference from the control group was
a decreased mean body weight gain in the 1000 ppm group males during study days 84-91. A similar reduction was not observed in the 2500 ppm group; therefore, this transient decrease was not attributed to the test article. No test article-related effects on maternal body weights or body weight gains were observed in the test article-exposed groups during the F0 gestation period. Differences from the control group were slight and were not statistically significant.
No test article-related effects on F0 lactation body weights or body weight gains were observed in the test article-exposed groups. The only statistically significant (p<0.01) differences from the control group were a mean body weight loss of 33 grams in the 500 ppm group compared to a loss of 53 grams in the control group during lactation days 21-28, resulting in a statistically significant (p<0.05 or p<0.01) increase in mean body weight on lactation day 28 and in mean body weight gain during the entire lactation period (days 1-28). Since the control group lost more weight than the 500 ppm group, this difference was not considered to be an adverse change.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
PARENTAL ANIMALS
Food consumption, evaluated as g/animal/day and g/kg/day, and food efficiency in the 500, 1000 and 2500 ppm groups were unaffected by exposure to the test article during the pre-mating period and one week following weaning (females) and throughout the study (males). Occasional statistically significant (p<0.05 or p<0.01) reductions in food consumption (primarily g/kg/day) were observed in the 2500 ppm group males compared to the control group values during study days 14-21 through 112-119. Food consumption (g/animal/day) in these males was generally not affected during these intervals. Mean body weights and food efficiency in the 2500 ppm group males and females were similar to or greater than the control values. Therefore, these sporadic reductions were not attributed to exposure to the test article. The only other statistically significant differences (p<0.05 or p<0.01) from the control group were a decrease in food consumption (g/animal/day) in the 2500 ppm group males during study days 28-35, an increase in food consumption (g/kg/day) during day 0-1 in the 1000 ppm group males and a decrease in food efficiency in the 1000 ppm group males during study days 1-4. Similar differences were not observed in the 2500 ppm group males during this interval; therefore, no relationship to exposure was evident.
F0 maternal food consumption and food efficiency during gestation were unaffected by test article exposure in the 500, 1000 and 2500 ppm groups. The only statistically significant difference from the control group was a slight reduction in food consumption (g/kg/day) in the 2500 ppm group when the entire gestation period (days 0-20) was evaluated. Food efficiency and gestation body weight gain in this group was unaffected during this interval. Therefore, the reduction was not attributed to test article exposure.
F0 maternal food consumption and food efficiency in the test article-exposed groups were similar to the control group values throughout lactation. The only statistically significant (p<0.01) differences were a smaller decrement in food efficiency in the 500 ppm group during lactation days 21-28 than the control group and an increase in food efficiency in this group during the entire lactation period (days 1-28). Because similar effects were not observed at higher exposure levels, these differences were not considered test article related.
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
PARENTAL ANIMALS
No test article-related effects on organ weights were observed in the 500, 1000 and 2500 ppm group males or females. The only statistically significant (p<0.05 or p<0.01) differences from the control group were increases in mean absolute prostate weights in the 1000 ppm group males and mean relative prostate weights in the 500 and 1000 ppm group males and a decrease in mean absolute pituitary weight in the 500 ppm group females. Because similar effects were not observed in the 2500 ppm group males and females, these changes were not attributed to the test article.
Gross pathological findings:
no effects observed
Description (incidence and severity):
PARENTAL ANIMALS
Female no. 74805 in the 500 ppm group was euthanized on lactation day 0 due to difficulties during parturition. This female delivered 12 pups and had four fetuses with no apparent malformations retained in utero. This animal had dark red contents in the ileum and jejunum and a pale pituitary gland. Similar findings were not observed in females at the scheduled necropsy in the higher exposure groups. Therefore, these findings were not attributed to the test article.
At the scheduled necropsy, no exposure-related trends were observed in the macroscopic findings noted in the test article-exposed groups. Findings were observed similarly in control group animals, were noted infrequently and/or did not occur in an exposure-related manner. The mean number of implantation sites and the mean number of unaccounted sites in the F0 test article-exposed females were similar to the control group values; no statistically significant differences were observed.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
PARENTAL ANIMALS
Female no. 74805 was euthanized in extremis during parturition. Microscopic findings for this female included severe acute renal tubular necrosis, moderate acute hepatic necrosis, lymphoid depletion of the spleen and lymphoid necrosis of the thymus. The cause of the moribund condition of this female was renal and liver necrosis with multiple organ failure.
At the scheduled necropsy, no test article-related microscopic findings were observed including for animals that failed to breed or produce a litter. The only statistically significant (p<0.05) differences from the control group values were an increase in the incidence of hydronephrosis in the 500 ppm group females and a decrease in the incidence of dilatation of the uterine lumen in the 2500 ppm group females. A slightly increased incidence (not statistically significant) of basophilic tubules was observed in the kidneys of the 2500 ppm group males; however, the severity of this lesion was minimal, and a similar increase was not observed in the 2500 ppm group females. Therefore, this common, spontaneous alteration was not considered to be test article-related. Other microscopic findings were typical of spontaneous conditions in young rats.
Histopathological findings: neoplastic:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Remarks:
parental, systemic
Effect level:
>= 7.5 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: original value: 2500 ppm; no effects observed

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion