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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 week
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted according to NTP standard study and GLP guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: NTP standard guideline for subchronic inhalation study
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-methylpropan-2-ol
EC Number:
200-889-7
EC Name:
2-methylpropan-2-ol
Cas Number:
75-65-0
Molecular formula:
C4H10O
IUPAC Name:
2-methylpropan-2-ol
Constituent 2
Reference substance name:
tertiary butyl alcohol
IUPAC Name:
tertiary butyl alcohol
Details on test material:
-Name of test material (as cited in study report): t-butyl alcohol
-Source of test material: FBC Chemical Corporation (Lancaster, NY)
-Analytical purity: >99% for each lot
-Appearance: clear, colorless liquid
-Lot No.: Un-1120 was used for part of the 13-week studies; Lots F0020487 and 07098 were used for the remainder of the 13-week studies
-Methods of analyses: all lots of the chemical were identified by IR, UV-VIS and NMR spectroscopy; purity determined by elemental analyses, Karl Fischer water analysis, and gas chromatography (GC). GC of lots UN-1120 and F020487 indicated one major peak and one impurity with a peak area greater than or equal to 0.1% relative to the major peak; GC of lot 070981 indicated two impurities with a combined peak area of 0.291 relative to the major peak. All spectra were consistent with those expected for tertiary butyl alcoho; IR and NMR spectra were consistent with literature spectra.
-Stability under test conditions: Identity, purity and stability analyses were conducted by the analytical chemistry laboratory Midwest Research Institute (Kansas City, MO). An accelerated stability study performed by the analytical chemistry laboratory indicated that, as a bulk chemical, tertiary butyl alcohol was stable when stored for 2 weeks protected from light at temperatures up to 60 °C.
-Storage condition of test material: the bulk chemical was stored at room temperature in amber glass bottles with Teflon-lined lids. Stability was monitored during the 13-week studies with gas chromatography. No degradation was detected.

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS:
-Source: Taconic Laboratory and Animal Services (Germantown, NY)
-Age at receipt: approx. 4 weeks; animals were held for 13 days before study initiation
-Age at study initiation: 7 weeks
-Method of Animal Distribution: Animals were distributed randomly into groups of approximately equal initial mean body weight
-Housing: animals were housed in individual cages in the exposure chambers
-Diet: NIH-07 Open Formula Diet (Zeigler Brothers, Inc., Gardners, PA) in pellet form was available ad libitum except during exposure periods. Food was changed weekly.
-Water: city water (Columbus, OH) was available ad libitum

ENVIRONMENTAL CONDITIONS:
-Temperature (°C): 21° to 27.5°
-Relative Humidity: 28% to 76%
-Air changes per hour: 13-17
-Photoperiod (hrs dark/ hrs light): 12 hours light/dark

IN-LIFE DATES:
-Date of First Exposure: 4-5 March 1987
-Date of Last Exposure: 3-4 June 1987
-Date of Necropsy: 4-5 June 1987

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: filtered room air
Remarks on MMAD:
MMAD / GSD: No information
Details on inhalation exposure:
The test material was delivered by a positive displacement metering pump to a slightly heated Sonimist Ultrasonic Spray Nozzle nebulizer. Liquid droplets 0.1 to 50 μm in diameter were discharged into a plenum chamber and evaporated in a stream of filtered, compressed air pumped through the chamber at 50 psig. To prevent solidification of the test material, all tubing, lines, and connectors were made of stainless steel or Teflon® and were slightly heated. Vapor was drawn into a common distribution manifold and either passed undiluted into the highest exposure chamber or diluted with filtered room air to achieve the lower concentrations. Flow rate was controlled by calibrated flowmeters located in the exhaust line immediately downstream of the chamber. Three times per day the pressure differential between each exposure chamber and the room, the chamber temperature and relative humidity, and the flow through each of the calibrated dilution flowmeters were recorded. The exposure chambers had a volume of 2m3. In the 18-day range finding study used to establish exposure concentrations for the key 13-week study, target and actual concentrations were virtually identical. In the 13-week study, nominal chamber concentrations were approximately 20% higher than target concentrations. Since the study report used nominal concentrations throughout, this IUCLID document has also used nominal concentrations in reporting results, discussions, and conclusions.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Tertiary butyl alcoho vapor concentrations were measured by an infrared analyzer with absorbance at two different wavelengths. A wavelength of 3326 μm was used to monitor the two lowest exposure chambers and a wavelength of 8598 μm was used to monitor the three highest exposure chambers. Vapor concentration was measured hourly during exposure periods by means of a movable probe which sequentially sampled 12 points within each chamber to ensure vapor concentration homogeneity.

Vapor concentration in each chamber was demonstrated to be uniform and stable. A correction factor of 1.2 was applied to the recorded vapor concentrations for the 13-week study due to a discrepancy in the method of use of the infrared analyzer noted during the course of the study. Due to this correction, nominal chamber concentrations of tertiary butyl alcohol during the study were 0, 134, 272, 542, 1080 and 2101 ppm.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hr/day, 5 days/wk (excluding weekends and a holiday) for 13 weeks with at least 2 consecutive exposure days before sacrifice.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
134 +/- 5 ppm (113 ppm target)
Basis:
other: nominal average chamber concentration
Remarks:
Doses / Concentrations:
272 +/- 9 ppm (225 ppm target)
Basis:
other: nominal average chamber concentration
Remarks:
Doses / Concentrations:
542 +/- 15 ppm (450 ppm target)
Basis:
other: nominal average chamber concentration
Remarks:
Doses / Concentrations:
1080 +/- 25 ppm (900 ppm target)
Basis:
other: nominal average chamber concentration
Remarks:
Doses / Concentrations:
2101 +/- 44 ppm (1750 ppm target)
Basis:
other: nominal average chamber concentration
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
The concentrations used in this study were based on findings in an 18-day study previously conducted for the NTP.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes for mortality/morbidity
-Time schedule: twice daily, 7 days per week

DETAILED CLINICAL OBSERVATIONS: Yes
-Time schedule: weekly

BODY WEIGHT: Yes
-Recorded prior to first exposure, weekly thereafter, and at the end of study

FOOD/WATER CONSUMPTION: no data

OPTHALMOSCOPIC EXAMINATION: not examined

HEMATOLOGY:
-Time schedule for collection of blood: collected at the end of the study.
-Number of animals: all mice/sex/group
-Parameters examined: hematocrit, hemoglobin concentration, erythrocyte count, reticulocyte count, nucleated erythrocyte count, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, platelet count, and leukocyte count and differential.

CLINICAL CHEMISTRY: not examined

URINALYSIS: not examined

NEUROBEHAVIORAL EXAMINATION: not examined
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. Complete necropsies were performed on all animals; organs and tissues were examined for gross lesions and were fixed and preserved in 10% neutral buffered formalin; tissues to be examined microscopically were trimmed, embedded in paraffin, sectioned to a thickness of 4 to 6 μm, and stained with hematoxylin and eosin (H&E). Tissues and organs collected included:
-adrenal gland
-bone and marrow
-brain (three sections)
-clitoral gland
-esophagus
-gallbladder
-heart
-large intestine (cecum, colon, rectum)
-kidney
-larynx
-liver
-lung
-lymph nodes (bronchial, mandibular, mediastinal, and mesenteric)
-mammary gland
-nose
-ovary
-pancreas
-parathyroid gland
-pituitary gland
-preputial gland
-prostate gland
-salivary gland
-skin
-small intestine (duodenum, jejunum, ileum)
-spleen
-stomach (forestomach and glandular)
-testis (with epididymis and seminal vesicle)
-thymus
-thyroid gland
-trachea
-urinary bladder
-uterus

ORGAN WEIGHTS: Yes, the following organ weights were recorded at termination – brain, heart, right kidney, liver, lung, right testis and thymus

HISTOPATHOLOGY: Yes, complete examinations were performed on all animals in the 0 and 2101 ppm groups and gross lesions were examined in animals from all exposure groups. Tissues examined microscopically included those listed above under gross pathology.
Other examinations:
SPERM MORPHOLOGY/VAGINAL CYTOLOGY EVALUATION: Female mice were evaluated for necropsy body weight, relative frequency of estrous stages, and estrous cycle length. Male mice were evaluated for necropsy body weight, reproductive tissue weights, and epididymal spermatozoal data.

-Animals evaluated: animals in 0, 542, 1080 and 2101 ppm groups
-Protocol: NTP Sperm Motility Vaginal Cytology Evaluation
-Time schedule: For 7 consecutive days prior to sacrifice, the vaginal vaults of 10 female mice were lavaged, and the aspirated lavage fluid and cells stained with Toluidine Blue. Sperm motility was evaluated at necropsy.
-Number of animals: 10 females and 10 males per exposure group
-Method (females): Relative numbers of leukocytes, nucleated epithelial cells, and large squamous epithelial cells were determined and used to ascertain estrous cycle state (i.e., diestrus, proestrus, estrus, or metestrus).
-Method (males): The right testis and epididymis were weighed. The tail of the epididymis (cauda epididymis) was removed from the epididymal body (corpus epididymis) and weighed. Modified Tyrode's buffer was applied to slides, and a small incision was made at the distal border of the cauda epididymis. The sperm effluxing from the incision were dispersed in the buffer on the slides, and the numbers of motile and nonmotile spermatozoa were counted for 5 microscopic fields per slide by two independent observers. Then each cauda epididymis was placed in phosphate-buffered saline solution. Caudae were finely minced and the tissue incubated and then heat fixed at 65 °C. Sperm density was determined microscopically and slides were prepared and stained with eosin for evaluation of sperm morphology.
Statistics:
Analysis of Continuous Variables:
Organ and body weight data were analyzed using the parametric multiple comparisons procedures of Williams (1971, 1972) or Dunnett (1955). Hematology, spermatid, and epididymal spermatozoal data were analyzed using the nonparametric multiple comparisons methods of Shirley (1977) or Dunn (1964). Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of dose-response trends and to determine whether a trend-sensitive test (Williams, Shirley) was more appropriate for pairwise comparisons than a test capable of detecting departures from monotonic dose response (Dunnett, Dunn). If the P-value from Jonckheere’s test was greater than or equal to 0.10, Dunn or Dunnett’s test was used rather than Shirley or Williams’s test.

The outlier test of Dixon and Massey (1951) was used to detect extreme values. No value selected by the outlier test was eliminated unless it was at least twice the next largest value or at most half of the next smallest value. The extreme values chosen by the statistical test were subject to approval by NTP personnel. In addition, values indicated by the laboratory report as being inadequate due to technical problems were eliminated from the analysis.

Analysis of Vaginal Cytology Data:
Because the data are proportions (the proportion of the observation period that an animal was in a given estrous stage), an arcsine transformation was used to bring the data into closer conformance with normality assumptions. Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for the simultaneous equality of measurements across exposure levels.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
Clinical signs and mortality: Five males exposed to 1080 ppm died during weeks 3 and 4; deaths occurred in animals in adjacent cages and were thought to be due to a water or feed availability problem. Feed and water consumption data were not available to confirm this. The death of one 2101 ppm male during week 7 was attributed to tertiary butyl alcoho exposure. All other animals survived to study termination. Clincal signs occurrred sporadically and were not considered related to chemical administration.

Body weight and weight gain: Initial mean body weight of 272 ppm males was significantly greater (8.6%) than controls, but mean body weight gain was significantly lower (27.1%). Final body weights relative to controls were 96-104% for male exposure groups. Mean body weight gains of 1080 and 2101 ppm females were decreased 18.6% and 23.7%, respectively; final mean body weight of 2101 ppm females was 8.5% lower than controls.

Hematology: At week 13, the segmented neutrophil count of 2101 ppm male mice was three times greater than controls. Other changes in hematology parameters were minimal, did not demonstrate a treatment relationship, and were not considered to be related to chemical exposure.

Clinical chemistry: not evaluated

Urinalysis: not evaluated

Organ weights: Relative liver weights of 1080 and 2101 ppm female mice were increased 9.3% and 20.6%, respectively, over controls. Other statistically significant organ weight differences in the 2101 ppm females were considered to be related to the lower final mean body weights in this group. Statistically significant increases in absolute and relative lung weights in 134 ppm and 272 ppm males were not observed at other exposure levels.

Gross pathology: Macroscopic observations at necropsy did not reveal any alterations that were considered treatment-related.

Histopathology: Microscopic observations at necropsy did not reveal any alterations that were considered treatment-related. No lesions corresponding to the increased liver weights were found. No lesions to account for early deaths were present.

Histopathology: neoplastic Not applicable

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 080 ppm
Sex:
male
Basis for effect level:
other: Based on a marked increase in segmented neutrophil count and a single chemically-related death in the 2101 ppm male exposure group, the NOAEL in male mice is considered to be 1080 ppm.
Dose descriptor:
NOAEL
Effect level:
2 101 ppm
Sex:
female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

No significant differences occurred in the reproductive endpoints of  males (weight of testis, epididymis, and cauda; sperm motility, count, and morphology) or females (estrous cycle length or percentage of time spent in the various estrous stages) exposed to 542, 1080 or 2101 ppm tertiary butyl alcohol.

Applicant's summary and conclusion

Conclusions:
The NOAEL following inhalation exposure of mice to tertiary butyl alcohol for 6 hr/day, 5 days/wk for 13 weeks was 2101 ppm in female mice and 1080 ppm in male mice.

There were no significant treatment-related effects on hematology or gross or microscopic effects on any target organ in female mice exposed to 2101 ppm tertiary butyl alcohol, the highest concentration tested. The only significant effect noted in male mice was a marked increase in segmented neutrophil count in the 2101 ppm exposure group only. Based on this information, tertiary butyl alcohol is not classified in mice exposed by the inhalation route for “Specific Target Organ Toxicity – Repeated Exposure” according to the GHS guidelines.
Executive summary:

In a subchronic toxicity study, tertiary butyl alcohol was administered to 10 B6C3F1 mice/sex/group by inhalation exposure at 0, 134, 272, 542, 1080 and 2101 ppm for 6 hr/day, 5 days/wk for 13 weeks. Based on a single compound-related death and a marked increase in segmented neutrophil count, the NOAEL in male mice is considered to be 1080 ppm.  Other than a decrease in final body weight and increase in relative liver weights, no effects were observed in female mice. In the absence of mortality, clinical signs, hematological effects or gross/microscopic effects, the NOAEL in female mice is considered to be 2101 ppm. Nephropathy observed in similarly exposed male rats was not observed in either sex of mice. Tertiary butyl alcohol had no adverse effects on reproductive endpoints in either sex.