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Repeated dose toxicity: inhalation

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Administrative data

sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study was conducted according to NTP standard and GLP guidelines.

Data source

Reference Type:
study report

Materials and methods

Principles of method if other than guideline:
Groups of 10 male and 10 female rats were exposed to t-butyl alcohol by inhalation at different concentrations for 6 hours per day, 5 days per week (excluding weekends and a holiday), for 13 weeks, with at least 2 consecutive exposure days before sacrifice.
GLP compliance:
Limit test:

Test material

Details on test material:
- Name of test material (as cited in study report): t-Butyl alcohol
- Physical state: clear colorless liquid
- Analytical purity: > 99 %
- Lot/batch No.: UN-1120, F020487 and 070981
- Stability under test conditions: An accelerated stability study performed by the analytical chemistry laboratory using gas chromatography showed that t-butyl alcohol was stable as bulk chemical for 2 weeks when it was stored 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 to ensure stability.
- Supplier: FBC Chemical Corporation, Lancaster, NY, USA

Test animals

other: F344/N
Details on test animals or test system and environmental conditions:
- Source: Taconic Laboratory and Animal Services (Germantown, NY)
- Age at study initiation: 7 weeks
- Weight at study initiation: male: 128.5 g; female: 110.5 g
- Housing: single housing
- Diet (e.g. ad libitum): NIH-07 Open Formula Diet (Zeigler Brothers, Inc., Gardners, PA, USA); ad libitum
- Water (e.g. ad libitum): City water (Columbus, OH, USA); ad libitum
- Acclimation period: 13 days

- Temperature (°C): 23.6 - 27.9 °C
- Humidity (%): 47 - 76 %
- Air changes (per hr): 13-17/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Details on inhalation exposure:

Liquid t-butyl alcohol was directed by a positive displacement metering pump to a slightly heated Sonimist Ultrasonic Spray Nozzle nebulizer. Liquid droplets with a diameter of 0.1 to 50 μm were discharged from the nebulizer into a plenum chamber and were evaporated in a stream of filtered, compressed air pumped through the chamber at 50 psig. All tubing, lines, and connectors were of stainless steel or Teflon® and were slightly heated to prevent solidification of the t-butyl alcohol. The vapor was drawn into a common distribution manifold and passed undiluted into the first exposure chamber or it was diluted with filtered room air by means of calibrated dilution flow meters to achieve the desired diluted concentrations for the other chambers. The flow rate through the chamber was controlled by calibrated flowmeters located in the exhaust line immediately downstream of the chamber. The following parameters were recorded three times daily: 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. The H-2000 exposure chambers had a 2 m3 volume.
The vapor concentrations of the test substance were measured by a infrared analyzer with absorbance at two different wavelengths: A wavelength of 3,326 um was used to monitor the three lowest exposure chambers. A wavelength of 8,598 um was used to monitor the three highest chambers. Vapor concentration was determined hourly during exposure periods by means of a movable probe which sequentially sampled 12 points within each chamber to ensure vapor concentration homogeneity. The nominal chamber concentrations of t-butyl alcohol were 0, 135, 270, 540, 1080, and 2100 ppm.

Vapor concentration in each chamber were shown to be uniform and stable.

Cold trap samples were collected from the highest and lowest concentration chambers during the last hour of a 6-hour exposure period in order to identify possible degradation products caused by vaporization. These samples were analyzed by gas chromatography-mass spectrometry. This analysis was performed with and without animals in the chambers. No degradation products were detected at concentrations equal to or exceeding 1% of the t-butyl alcohol concentrations during the study. In addition, the exposure chambers, the control chamber, and the room air were monitored with an aerosol monitor (APS-33, TSI, Minneapolis, MN, USA) both prior to and during the studies. Herewith, any particulate material that might arise during any part of the generation process could be detected. No significant particulate material was detected.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
t-Butyl alcohol vapor concentrations were quantified by an infrared analyzer with absorbance at two different wavelengths. A wavelength of 3,326 um was used to monitor the three lowest exposure chambers. A wavelength of 8,598 um was used to monitor the two highest exposure chambers. Vapor concentration was measured hourly during exposure periods by a movable probe which sequentially sampled 12 points within each chamber to ensure vapor concentration homogeneity.
During the study, buildup and decay rates for chamber concentrations were monitored using the Miran-80 infrared analyzer. Values were collected at 60-second intervals and plotted by computer. The time after the start of exposure required for the t-butyl alcohol concentration to reach 90% of the stable concentration level was identified as T90 for chamber wash-in, and the time following termination of vapor generation required for the aerosol concentration to decay to 10% of the stable concentration was identified as the T10 for chamber wash-out. The measured mean T90 wash-in value of 9 minutes was in agreement with the theoretical value of 9.2 minutes; a T90 of 10 minutes was used during the study.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 h/d; 5d/w
Doses / concentrationsopen allclose all
Doses / Concentrations:
0, 113, 225, 450, 900, 1750 ppm = 0, 0.34, 0.68, 1.3, 2.7, 5.3 mg/L
nominal conc.
Doses / Concentrations:
0, 135, 270, 540, 1080, and 2100 ppm = 0, 0.4, 0.8, 1.6, 3.2, 6.4 mg/L
analytical conc.
No. of animals per sex per dose:
10 animals/sex and dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Concentrations were based on the 18-day dose-finding study


Observations and examinations performed and frequency:
- Time schedule: twice daily for 7 days/week
- Cage side observations checked: Mortality and morbidity

- Time schedule: weekly

- Time schedule for examinations: prior to the first exposure, weekly thereafter, and at the end of the study

- Body weight gain: Yes

- Time schedule for collection of blood: day 22 and at the end of the study
- Anaesthetic used for blood collection: Yes (with a mixture of carbon dioxide and oxygen)
- How many animals: all animals
- Parameters that were 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.

- Time schedule for collection of blood: day 22 and at the end of the study
- How many animals: all animals
- Parameters checked: alanine aminotransferase, alkaline phosphatase, sorbitol dehydrogenase, y-glutamyltransferase, and bile salts

- Time schedule for collection of urine: day 21 and at the end of the study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes but water was available
- Parameters checked: urine volume, specific gravity, pH, and microscopic examination of sediment

OTHER: Organ weights for brain, heart, right kidney, liver, lung, right testis, and thymus were determined. Tissues that were examined microscopically included: adrenal gland, bone and marrow, brain, clitoral gland, esophagus, 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 and the kidneys of all groups of male animals. Gross lesions were examined microscopically in all exposure groups.
In addition, in the 0, 540, 1080, and 2100 ppm groups, animals were evaluated for body weights, reproductive tissue weights (right cauda, right epididymis, and right testis) and epididymal spermatozoal data (sperm density, morphology, and motility). Female rats were evaluated for necropsy body weight, relative frequency of estrous stages, and estrous cycle length.
Sacrifice and pathology:
Two approaches were employed to determine the significance of pairwise comparisons between exposed and control groups in the analysis of continuous variables. Data received from organs and body weights were analyzed using the parametric multiple comparisons procedures of Williams (1971, 1972) or Dunnett (1955).
Jonckheere's test (Jonckheere, 1954) was used to determine the significance of dose-response trends and to ascertain 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). In case the P-value from Jonckheere's test was greater than or equal to 0.10, Dunn's or Dunnett's test was used rather than Shirley's or Williams' test.
The outlier test of Dixon and Massey (1951) was used to detect extreme values. Outliers were eliminated when they were at least twice the next largest value or at most half of the next smallest value. Values rated by the laboratory report as being inadequate due to technical problems were eliminated from the analysis.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY: one male animal was killed accidentally during blood collection on day 22, all other animals survived

BODY WEIGHT AND WEIGHT GAIN: body weight gain was similar in all groups, no findings could be observed

HAEMATOLOGY: A decrease in hematocrit values, hemoglobin concentrations, and erythrocyte counts occurred at the applied doses of 1080 and 2100 ppm in male rats. Lower doses also resulted in significantly decreased hemoglobin values in male rats but failed to be dose-dependent. The cell hemoglobin concentration, reticulocyte and nucleated erythrocyte counts were not altered.

CLINICAL CHEMISTRY: A significant decrease in alkaline phosphatase activity occurred in the following dose groups: 1080, and 2100 ppm in male rats. A dose-dependent decrease was already shown in the 540 ppm dose group but failed to be significant; no other treatment-related findings could be determined.

URINALYSIS: A decrease in urine pH occurred at a concentration of 2100 ppm in males and at 1080 and 2100 ppm in females. Other occurring changes in clinical pathology variables were minimal and were not considered to be related to chemical exposure.

ORGAN WEIGHTS: Absolute and relative right kidney weights of males receiving 1080 and 2100 ppm were significantly increased compared to the controls. The relative right kidney weight of females receiving 2100 ppm and the relative liver weights in females receiving 1080 and 2100 ppm were significantly greater than those of the controls. Other statistically significant differences in organ weights of males were not considered related to chemical administration.

GROSS PATHOLOGY: No treatment-related findings could be observed.

HISTOPATHOLOGY: NON-NEOPLASTIC: In male rats, a treatment-related increase in the severity of chronic nephropathy relative to the controls occurred in all exposed groups as evidenced by an increased number of foci per section. Sections of kidneys from male animals in the 0, 1080, and 2100 ppm groups (four per group) were stained by the Mallory-Heidenhain method for the analysis of tubular hyaline droplet accumulation. No difference between control and exposed animals in the number, size, or shape of renal tubule hyaline droplets could be detected.

OTHER FINDINGS: There were no significant effects in reproductive tissue parameters or estrous cycle characterization between exposed and control groups

Effect levels

open allclose all
Dose descriptor:
Effect level:
ca. 135 ppm
Based on:
test mat.
Basis for effect level:
other: kidney effects (weights and severity of nephropathy increased)
Dose descriptor:
Effect level:
ca. 540 ppm
Based on:
test mat.
Basis for effect level:
other: increased kidney and liver weights, reduced pH-values
Dose descriptor:
Effect level:
ca. 270 ppm
Based on:
test mat.
Basis for effect level:
other: Clinical chemistry and decreased hematology values

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion