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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: GLP guideline study conducted in compliance with OPPTS Guideline 870.3465.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006
Reference Type:
publication
Title:
Unnamed
Year:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tertiary Butyl Acetate
- Source of test material: Lyondell Chemical Company, Deer Park, TX
- Date of receipt: 23 May 2005
- Expiration Date: 1 May 2008
- Appearance: clear, colorless liquid
- Analytical purity (when packaged): 99.68%
- Analytical purity (during testing): 99.51-99.63%
- Lyondell Material: 499245 TBAc TM
- Lot/batch No.: OD50502001
- Stability under test conditions: stable
- Storage condition of test material: Refrigerated in sponsor supplied drums

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS:
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina
- Number of animals: 140 males and 140 females
- Age at receipt: 35 days
- Age at study initiation: approx. 7 weeks
- Weight at study initiation: 27.4-33.4 g for males and 21.2-27.5 g for females
- Housing: Upon arrival, weanling animals were housed 3 per cage (or 2 per cage, if necessary) by sex in clean, suspended, stainless steel wire-mesh cages for at least 3 days following receipt. Thereafter, all animals were housed individually in clean, suspended, stainless steel wire-mesh cages in an environmentally controlled room.
- Diet: PMI Nutrition International, LLC Certified Rodent Lab Diet® 5002 (meal) ad libitum except during exposures
- Water: Reverse osmosis-treated tap water available ad libitum
- Acclimation period: 16 days
- Identification method: tail tattoo
- Method of animal distribution: Animals were weighed and examined for physical abnormalities; data were collected using the WIL Toxicology Data Management System. Animals were divided into groups of 30 males and 30 females.


ENVIRONMENTAL CONDITIONS:
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20
- Air changes (per hr): 10 room air changes per hour, 100% fresh air except during exposure. Exposure chambers were operated under dynamic conditions at a slight negative pressure with at least 12 to 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours light/dark

IN-LIFE DATES:
9/27/2005 (animal receipt)
01/12-13/2006 (primary necropsy)

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: filtered air
Details on inhalation exposure:
Exposures were conducted in four 2.0 m3 stainless steel and glass exposure chambers. Three chambers were dedicated for the test substance exposure groups, while the fourth chamber was dedicated for the filtered air control group. All chambers were operated under dynamic conditions at a slight negative pressure with at least 12 to 15 air changes per hour. A HEPA filter and an activated charcoal bed were used to treat chamber air supply. Animals in each exposure group were caged individually. The cage batteries were rotated on a daily basis within the chambers to ensure similar exposure to all animals over the duration of the study. For each exposure, the animals were transferred from their home cages to the whole-body exposure cages, transported to the exposure room, exposed for the requisite duration, and returned to their home cages. Food was withheld during exposure but water was available ad libitum.

Tertiary butyl acetate was generated for exposure as a vapor using a glass-bead, column-type vaporization system. The liquid test substance was metered to the column using a laboratory pump and nitrogen carrier gas was metered to the bottom of the column. The column was heated to an appropriate temperature to produce vaporization of the test substance. The concentrated vapors were piped to the chamber inlet where the concentration was reduced by mixing with chamber ventilation air.

Filtered-air and test substance vapor were administered as six-hour, whole-body exposures for 13 weeks with exposures on 7 days per week to 30 animals/sex/group at target exposure levels of 0, 100, 400 and 1600 ppm. Temperature, relative humidity, chamber ventilation rate and negative pressure within the exposure chambers were monitored continuously and recorded approximately every 35 minutes. All animals were exposed at approximately the same time each day. Males and females were not exposed on Study Days 35 and 36 due to blood collection. Ten animals/sex/group were used for blood collection for clinical pathology evaluations (and then euthanized and discarded) after receiving at least 28 exposures (not more than 32 exposures). Blood was collected from up to 10 animals/sex/group assigned to the interim blood collection for assessment of the hormonal status of the pituitary-thyroid axis. Following blood collection, these animals were also euthanized and discarded. On the day following the final exposure (by sex), surviving animals were euthanized and necropsied. The first day of exposure is defined as exposure Day 0 (Study Day 0) and the first week of exposure is defined as Study Week 0.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Nominal Concentrations:
A nominal exposure concentration was calculated for each daily exposure for each chamber from the total amount of tertiary butyl acetate used during the exposure and the total volume of air passed through the chamber during that day's exposure. The amount of test substance used was obtained by weighing the reservoir containing the test substance for each chamber prior to and after each day's exposure. The total volume of air passed through each chamber was calculated from the daily average chamber ventilation flow rate in liters per minute (LPM) and the exposure duration.

Analyzed Exposure Concentrations:
Analyzed exposure concentrations were determined at least 10 times during each 6-hour exposure using a gas chromatographic (GC) method with flame ionization detection. Samples of the exposure atmospheres were automatically collected from the approximate animal-breathing zone of the inhalation exposure chambers at approximately 35-minute intervals using a sample loop and a computer-controlled multi-position valve. A natural log quadratic equation based on the GC calibration curve was used to calculate the measured concentrations in parts per million (ppm). The overall mean analyzed concentrations were 0, 101, 400 and 1598 ppm. Because the analyzed concentrations were in good agreement with the target concentrations and to allow easier comparison between studies done in different species over different exposure periods, target rather than analyzed concentrations will be used for reporting and discussion purposes in this submission.

Purity Analysis:
A sample of the test substance was collected from each primary test substance container prior to initial use and near the end of use. Vapor generator reservoir stability was evaluated using the same analysis methods and analyzing samples collected from a generator reservoir at the beginning and end of a 7-day generation period. The percent concentration of tertiary butyl acetate was determined by the Analytical Chemistry Department of WIL Research Laboratories using a gas chromatographic method with detection by flame ionization for each sample. Purity of tertiary butyl acetate was expressed as area percent.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hrs/day 7 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
101 ppm
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
400 ppm
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
1598 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
30 animals/sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale:
The exposure levels were based on the results of a 14-day range-finding study.

Rationale for animal assignment:
At the conclusion of the acclimation period, animals judged to be suitable for testing were randomly assigned to groups based on body weight. Individual body weights were within +/- 20% of the mean for each sex and there were no statistically significant differences among the groups (i.e., p-value for ANOVA not significant at p < 0.05).

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes for mortality/viability
-Time schedule: twice daily (7 hours apart)

CLINICAL OBSERVATIONS: Yes, clinical observations were conducted prior to exposure and 0-1 hour following the exposure on each exposure day. The clinical observations prior to exposure were not performed on the days of detailed physical examinations. At approximately the midpoint of each 6-hour exposure, animals that were visible through the chamber windows were observed for appearance and behavior.

DETAILED PHYSICAL EXAMINATIONS: Yes, all animals received a detailed physical examination weekly, beginning at least one week prior to test substance administration (to include examination for randomization) and continuing through (and including) the day of necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded weekly, beginning 3 weeks prior to exposure (Study Week -3). Body weights were recorded prior to first exposure and prior to scheduled necropsy.

FOOD CONSUMPTION: Yes
- determined weekly; beginning approximately 2 weeks prior to first exposure.

ESTROUS CYCLE DETERMINATION: Yes
- Time schedule for examinations: After 10 weeks of exposure, the stage of estrus cycle was determined daily for 3 weeks in approximately 10 female animals/group.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: both eyes were examined prior to first exposure during Study Week -1 (30 animals/sex/group) and at 12 weeks (approximately 10 animals/sex/group)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Study Week 4, 5 and 13
- Anaesthetic used for blood collection: Yes (isoflurane)
- Number of animals: approximately 10 animals/sex/group
- Collection of sample via: retro-orbital sinus

Blood was collected into tubes containing EDTA (hematology) or no anticoagulant (serum chemistry). Due to irregularities in the hematology data from samples collected during Study Week 4 (likely caused by low sample volumes), interim hematology evaluations were repeated during Study Week 5 on the remaining animals designated for evaluation following 13 weeks of exposure. Blood volume during Study Week 5 was limited to 0.3 mL based on mean body weights for survival following blood collection. The hematology results from the Study Week 4 blood collection were considered invalid, and only the Study Week 5 interim hematology results were reported. The Study Week 4 hematology results were maintained in the study records.

- Parameters listed below were examined:
Hemoglobin, hematocrit, erythrocyte count, total leukocyte count, differential leukocyte count, platelet count, reticulocyte counts, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC).


BLOOD CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Study Week 4, 5 and 13
- Anaesthetic used for blood collection: Yes (isoflurane)
- Number of animals: approximately 10 animals/sex/group/assessment
- Collection of sample via: retro-orbital sinus

- Parameters listed below were examined (blood chemistry):
Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, sorbitol dehydrogenase, phosphorus, glucose, urea nitrogen, creatinine, triglycerides, total cholesterol, total bilirubin, total protein, albumin, globulin, A/G ratio, calcium, chloride, potassium, sodium.

HORMONE EVALUATION: Yes
- Time schedule for collection of blood: Study Week 4
- Anaesthetic used for blood collection: Yes (isoflurane)
- Number of animals: approximately 10 animals/sex/group/assessment
- Collection of sample via: retro-orbital sinus

-Parameters listed below were examined (thyroid hormone assessment):
thyroid stimulating hormone (TSH), triiodothyronine (T3) and thyroxine (T4).

NEUROBEHAVIOURAL EXAMINATION: Yes
A modified FOB was performed on all remaining animals.
- Time schedule: Day 62 (males) and Day 63 (females); beginning about 15 minutes and completed within approximately 2.5 hours after the 6 hour inhalation exposure
- Number of animals: approximately 10 animals/sex/group
- Parameters examined: ease of removal from cage, ease of handling animal, salivation, lacrimation/chromodacryorrhea, palpebral closure, piloerection, red/crusty deposits, mucous membranes/eye/skin color, fur appearance, eye prominence, respiratory rate/character, muscle tone, mobility, gait, convulsions/tremors, arousal, bizarre/stereotypic behavior, rearing, backing, grooming, startle response, tail pinch response, righting reflex, forelimb grip strength, body temperature.
Sacrifice and pathology:
GROSS PATHOLOGY: A complete necropsy was conducted on all animals dying spontaneously, euthanized in extremis or at the scheduled necropsy. Macroscopic examination included examination of the external surface, all orifices and the cranial, thoracic, abdominal and pelvic cavities including viscera. At the time of necropsy, the following tissues and organs were collected and placed in 10% neutral-buffered formalin (except as noted); complete liver (with gallbladder) and a piece of duodenum were transferred into 70% ethanol after approximately 2 days in the formalin:

Adrenals (2), aorta, bone with marrow (sternum, femur), bone marrow smear (a), brain (cerebrum Level 1, cerebrum Level 2, cerebellum with pons/medulla), epididymides (2)b, eyes with optic nerves (2)c, gallbladder, gastrointestinal tract (esophagus, stomach, duodenum, jejunum, ileum with Peyer’s patches, cecum, colon, rectum), Harderian glands (2), heart, kidneys (2) #, lacrimal glands [exorbital(2)], larynx, liver #, lungs (including bronchi, fixed by, inflation with fixative) #, lymph node (mediastinal and bronchial [if visible], mesenteric), mammary gland (females only), nasal cavity (d), ovaries/oviducts (2) #, pancreas, peripheral nerve (sciatic), pharynx, pituitary, prostate, salivary glands [mandibular(2)], seminal vesicles (2), skeletal muscle (rectus femoris), skin, spinal cord (cervical, midthoracic, lumbar), spleen, testes (2)b, thymus, thyroid gland [with parathyroids (2)]e #, trachea, urinary bladder, uterus with cervix, vagina, all gross lesions .

a = Bone marrow smears were obtained at scheduled necropsy and from all animals euthanized in extremis (not placed in formalin).
b = Placed in Bouin’s solution.
c = Placed in Davidson’s solution.
d = Following collection of the appropriate protocol-specified tissues, the entire head was removed and preserved. Following decalcification, four cross-sections of the nasal cavities were prepared for microscopic examination in accordance with the method described by Young (1981).
e = Parathyroids were examined microscopically if in the plane of section and in all cases when a gross lesion of the parathyroid was present.

ORGAN WEIGHTS: Yes, the following organ weights were recorded at termination:
Brain, epididymides, kidneys, liver, lungs, ovaries/oviducts, spleen, thyroid with parathyroids (if present), thymus, testes, uterus.

HISTOPATHOLOGY: see list above under gross pathology - taken from control and 1600 ppm animals except for organs marked with # (all groups examined).

Reference:
Young JT, 1981. Histopathological examination of the rat nasal cavity. Fundamental and Applied Toxicology, 1:309-312.
Other examinations:
LIVER CELL PROLIFERATION:
Liver cell proliferation was assessed using immunohistochemical staining for proliferating cell nuclear antigen (PCNA). For each animal from the scheduled terminal necropsy, one paraffin section was stained for PCNA and examined to provide an index of cell proliferation.
Statistics:
All analyses were performed using two-tailed tests (except as noted otherwise) for a minimum significance level of 5% and performed separately by sex. All means were presented with standard deviations and the number of animals (N) used to calculate the mean. All statistical tests were performed by a computer with appropriate programming.

Continuous data variables (including mean body weights, body weight gains, food consumption at each interval, estrous cycle length, clinical pathology parameters and organ weights) were analyzed for heterogeneity of variance using Levene’s test (Levene, 1960) and for normality using the Shapiro-Wilk test (Royston, 1982). If the data were homogeneous and normal, a parametric one-way analysis of variance (ANOVA) was used to determine intergroup difference (Snedecor and Cochran, 1980). If the results of the ANOVA were significant (p < 0.05), Dunnett's test was applied to the data to compare the treated groups to the control group (Dunnett, 1964). If the data were not homogeneous and normal, a Kruskal-Wallis nonparametric ANOVA was used to determine intergroup difference (Kruskal and Wallis, 1952). If the results of the ANOVA were significant (p < 0.05), the Dunn’s Test was applied to the data to compare the treated groups to the control group (Dunn, 1964). See references below.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL OBSERVATIONS/MORTALITY:
There were no test substance-related unscheduled deaths during the course of the study. Two females from the control group, 2 males from the 100 ppm group, 1 male and 1 female from the 400 ppm group, and 1 male and 1 female from the 1600 ppm group were found dead or euthanized in extremis before completion of the exposure period. The early mortality was considered incidental based on the lack of dose response. All other animals survived to the scheduled necropsy.

Clinical findings that were considered treatment-related in 1 or more test substance groups consisted of hyperactivity and excessive grooming at the 1-hr post-exposure time point (observed in an exposure concentration-related manner for occurrence and total number of animals affected in 400 and 1600 ppm males and females) and impaired equilibrium (increased in the 400 and 1600 ppm females and possibly the 1600 ppm males) and labored respiration (increased in the 1600 ppm males and females) at the exposure midpoint. These clinical findings were acute and transient effects of daily exposure, with no test substance-related findings being present at the pre-exposure observation sessions.

BODY WEIGHT AND WEIGHT GAIN:
There were no test substance-related effects on body weights. Mean body weight gains and cumulative body weight gains were occasionally statistically significant in the test substance-treated groups compared to the control group. However, these differences were not observed in a dose-related manner and/or were transient in nature.

FOOD CONSUMPTION:
There were no test substance-related effects on food consumption although normal fluctuations in food consumption were noted. There were no consistent statistically significant differences when the control and test substance-treated groups were compared.

ESTROUS CYCLE DETERMINATION:
Estrous cycle lengths were unaffected by test substance administration. There were no statistically significant differences when the test substance-exposed females were compared to the control group.

OPHTHALMOSCOPIC EXAMINATION:
No ophthalmic lesions indicative of toxicity were observed in any of the test substance-exposed groups. All findings observed were typical in prevalence and appearance for laboratory mice of this age and strain.

HAEMATOLOGY:
There were no clear test substance-related changes in hematology and coagulation (platelet count only) parameters. Some small statistically significant (p < 0.05) differences were observed when the control and test substance-exposed groups were compared in female animals exposed to 1600 ppm (hemoglobin and hematocrit). These group mean differences were considered either spurious (not test substance-related) or not toxicologically significant because the individual values from the 1600 ppm group were within the concurrent control group values ± 10% and the differences from control means were less than ± 10%. In addition, the slightly higher mean hemoglobin and hematocrit values for females (Week 5) and mean hemoglobin values in males (Weeks 5 and 13) were not accompanied by changes in red blood cell numbers or in the RBC indices (mean corpuscular volume, mean corpuscular hemoglobin or mean corpuscular hemoglobin concentration).

SERUM CHEMISTRY:
There were no clear test substance-related changes in serum chemistry parameters. Some small statistically significant (p < 0.05) differences were observed when the control and test substance-exposed groups were compared (increased calcium during Study Week 4 in females exposed to 1600 ppm, and increased calcium and decreased chloride in males during Study Week 13 exposed to 1600 ppm, and increased phosphorus in males during Study Week 4 exposed to 100 ppm). These group mean differences were considered either spurious (not test substance-related) or not toxicologically significant because the values did not show an exposure concentration- or time-related response, individual values from the test substance exposed group were within the concurrent control range ± 10% and differences from control means were small in magnitude. Although higher mean calcium levels were observed for both males and females (9.9 mg/dL [Week 13] and 10.7 mg/dL [Week 4], respectively), these mean values were well within 2 standard deviations (SDs) from the WIL Research Laboratories’ historical control mean values (mean ± SD: 9.9 ± 0.6 and 10.1 ± 0.5 for males and females, respectively).

HORMONE EVALUATION:
Test substance-related lower T4 levels were found for male mice. Lower mean total T4 levels were statistically significant (p < 0.05) in males at 1600 ppm. Although there appeared to be dose-related lower mean T4 levels in males, there were no differences for mean TSH or T3 levels and there were no test substance-related histopathologic changes in the thyroid gland. The test substance-related lower circulating T4 levels may be related to hepatic metabolizing enzyme induction with higher turnover of T4 since the liver is the primary site of both deiodination of T4 to produce active T3 and inactive reverse-T3 and T2 and enzymatic conjugation of T4 to glucuronides and sulfates that are excreted. Increased liver weight without findings indicative of liver injury, which was observed in males and females exposed to 1600 ppm, is consistent with the presence of metabolizing enzyme induction.

NEUROBEHAVIOURAL EXAMINATION:
Modified functional observational battery parameters evaluated on Study Day 62 (males) and Study Day 63 (females) were not affected by exposure to tertiary butyl acetate. There were no significant differences between the control and test substance-exposed groups when observations were evaluated 1 to 2 hours following exposures on Study Days 62 (males) and 63 (females).

GROSS PATHOLOGY:
There were no test substance-related gross necropsy observations.

ORGAN WEIGHTS:
Test substance-related organ weight changes were limited to higher liver weights in males and females at 1600 ppm. The only histological correlate consisted of centrilobular hepatocellular hypertrophy in 1/10 females from the 1600 ppm group and very slightly higher PCNA labeling index in females at this exposure level. The following organ weight changes: kidney (relative to body and brain) in male mice in the 400 ppm group, lungs (relative to brain) and ovaries (relative to brain) in female mice in the 100 ppm group were statistically significant when compared to the control group, but the dose association was incoherent and no correlating microscopic findings were observed; thus the changes were considered to be spurious.

HISTOPATHOLOGY: NON-NEOPLASTIC:
Test substance-related changes were limited to minimal centrilobular hypertrophy in the liver of 1/10 females at 1600 ppm and correlated to the slightly higher liver weights at this exposure level. Degeneration of the X zone in the adrenal cortex was increased in incidence and severity in females at 1600 ppm compared to the control group. Despite the higher incidence and severity at 1600 ppm, the change was considered part of the normal degeneration process commonly noted in female mice as there were no morphological differences (except increased severity) between control and test substance-exposed animals. In all cases noted, the degeneration of the X zone was characterized by lipid vacuolation of the cortico-medullary junction. There were no other test substance-related histologic changes.

LIVER CELL PROLIFERATION
There was a very slightly higher number of PCNA-positive hepatocytes in the 1600 ppm females compared to the control group. The average number of PCNA positive hepatocytes in the 1600 ppm females was 4.3 for every 2000 hepatocytes (PCNA labeling index = 0.22%), compared to 2.1 for every 2000 hepatocytes in the control females (PCNA labeling index = 0.11%). The difference from control was not statistically significant.

Effect levels

open allclose all
Dose descriptor:
NOAEC
Effect level:
100 ppm
Sex:
male/female
Basis for effect level:
other: Based on transient acute clinical findings that were observed in the 400 and 1600 ppm tertiary butyl acetate-exposed groups during and following exposures.
Dose descriptor:
NOAEC
Effect level:
400 ppm
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Based on stability analyses from test substance storage containers and inhalation vapor generation reservoirs, the test substance was found to be stable when stored at room temperature in the sponsor-supplied drums over the duration of the study and was stable for at least 7 days when stored at room temperature in the inhalation atmosphere generator reservoir.

Applicant's summary and conclusion

Conclusions:
The NOAEC for systemic toxicity (non-acute) of tertiary butyl acetate, administered via whole body inhalation exposure 6 hours/day for 90 consecutive days, was 400 ppm for male and female CD-1 mice. The NOAEC for transient acute signs of toxicity in this study was 100 ppm. There were no test-substance related deaths and no significant treatment-related effects on body weight and body weight gain, food consumption, estrous cycle length, ophthalmic or functional observational battery parameters, hematology, serum clinical chemistries or gross pathology. Hyaline droplet nephropathy observed in the kidneys of male rats treated with the same exposure regimen was not observed in either sex of mouse. Test substance-related effects were limited to transient central nervous system effects in the 400 and 1600 ppm exposure groups that did not increase in severity or incidence in any consistent manner throughout the 13 weeks of exposure, slight liver enlargement in both sexes at 1600 ppm with minimal centrilobular hypertrophy in a single female, lower T4 levels (but no effects on TSH, T3, or microscopic evaluation of the thyroid) in 1600 ppm males, and a marginally higher PCNA labeling index in 1600 ppm females only. Based on this information, tertiary butyl acetate is not classified in mice exposed by the inhalation route for “Specific Target Organ Toxicity – Repeated Exposure” according to Directive 67/548/EEC, the UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS) or the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.
Executive summary:

In a subchronic toxicity study, tertiary butyl acetate was administered to 30 CD-1 mice/sex/group via whole-body inhalation at target exposure concentrations of 0, 100, 400 and 1600 ppm (actual concentrations were 0, 101, 400 and 1698 ppm) for 6 hr/day, 7 days/wk for 90 days. Test substance-related effects were limited to transient clinical observations in the 400 and 1600 ppm groups (hyperactivity and excessive grooming at post-exposure observations and impaired equilibrium and labored respiration [1600 ppm only] at the exposure midpoint) and slight liver enlargement in both sexes at 1600 ppm (as evidenced by higher liver weights) associated with minimal centrilobular hypertrophy in a single female and a marginally higher PCNA labeling index in 1600 ppm females only. Liver weight effects were more pronounced in female mice. In addition, lower statistically significant mean T4 levels were found in 1600 ppm group males.

 

In a 2-week inhalation toxicity study used to establish exposure levels for the 13-week study, higher liver weights were found for females and minimal centrilobular hypertrophy was observed in 2 of 5 males and 5 of 5 females exposed to 1500 ppm. The pattern of these liver findings, including the higher incidence of centrilobular hypertrophy in animals exposed to a similar concentration for a shorter duration, appears to represent metabolizing enzyme induction. Findings related to liver enzyme induction have been reported to be typically less pronounced following long-term repeated exposure than following short-term repeated exposure.

 

The NOAEC for acute effects in the 13-week study is considered to be 100 ppm based on transient clinical findings that were observed in the mid- and high-exposure groups during and following exposures while the NOAEC for systemic target organ effects was considered to be 400 ppm for male and female CD-1 mice based on minimal effects on the liver.