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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007
Reference Type:
publication
Title:
Unnamed
Year:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
. A number of specific observations known not to be sensitive to MTBE were excluded whilst others potentially sensitive were added.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): methyl tertiary-butyl ether (MTBE)
- Substance type: organic
- Physical state: liquid
- Analytical purity: >99.9%
- Lot/batch No.: 11333TD
- Stability under test conditions: stable
- Storage condition of test material: at room temperature
- Supplier: Sigma-Aldrich (St. Louis, MO)

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic (Germantown, NY)
- Strain: Hannover GALAS
- Age at study initiation: 4-6 weeks upon receipt, 6-8 weeks at the beginning of exposure
- Housing: inidvidually in polycarbonate cages with filter tops and on Alpha-Dri bedding. Cages cleaned weekly.
- Diet: NTP 2000 diet (zeigler Brothers Inc., Gardners, PA), ad libitum
- Water: reverse osmosis purified water, ad libitum
- Acclimation period: 12-14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 64-79 [18-26C]
- Humidity (%): 30-70%
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Fresh MTBE drinking water solutions were prepared and dispensed weekly. From each preparation event samples were retained to verify the concentration by GC analysis of headspace concentration. Aditionally at the end fo the week, samples were taken from selected water bottles to determine the concentration of the dosing solution after use by the animals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
From each preparation of the dosing solution, samples were retained to verify the concentration by GC analysis of headspace concentration. Airborne concentrations in exposure chambers were also monitored for 1 hour every 24 hours over a 6 day period.
Duration of treatment / exposure:
13 weeks (maximum of 93 days)
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.5, 3, 7.5 and 15 mg/ml
Basis:
nominal in water
Remarks:
Doses / Concentrations:
males: 37±10, 209±61, 514±142, 972±288 mg/kg bw/d
Basis:
other: average daily dose calculated from water consumption
Remarks:
Doses / Concentrations:
females: 50±12, 272±51, 650±142, 1153±191 mg/kg bw/d
Basis:
other: average daily dose calculated from water consumption.
No. of animals per sex per dose:
Control, 0.5 mg/ml, 3 mg/ml and 15 mg/ml: 20 in the core group, 20 in the clinical pathology subgroup, 30 in the cell replication subgroup
7.5 mg/ml: 20 in the core group, 20 in the clinical pathology group, 0 in the cell replication group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on available MTBE repeated dose studies (including 2-week pilot study and oral and inhalation exposure bioassays). The high dose was selected to approximate the high dose levels administered in previous studies; intermediate dose levels were set to be approximately 2 and 5-fold lower than the high dose. The low dose of 0.5 mg/ml was designed to be orders of magnitude greater than the known drinking water contaminant levels and to achieve a NOAEL.
- Rationale for animal assignment (if not random): based on body weight, using a weight randomization procedure in Provantis (v. 4.5, Instem, Conshohoken, PA)
- Rationale for selecting satellite groups: subgroups were formed to assess clinical pathology, cell replication and anatomic histopathology endpoints

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
Daily, including weekends and holidays, for mortality and overt clinical signs of toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed examination for clinical signs of disease or abnormality was performed once per week using the Provantis list of clinical observations

BODY WEIGHT: Yes
- Time schedule for examinations: every Monday, Tuesday, Wednesday, Thursday and Friday for control, 0.5 mg/ml, 3 mg/ml, 7.5 mg/ml and 15 mg/ml dose group animals, respectively. Each rat was weighed prior to study start, at the start of MTBE exposure, then once per week for 13 weeks. A final body weight was recorded prior to necropsy.

FOOD CONSUMPTION:
- Time schedule for examinations: every Monday, Tuesday, Wednesday, thursday and Friday for control, 0.5 mg/ml, 3 mg/ml, 7.5 mg/ml and 15 mg/ml dose group animals, respectively. Food consumption was measured gravimetrically, per animal, for 13 weekly intervals, and reported as g/animal/day. Final data was normalized to the body weight by diving the weight of food consumed by the mean of the interval's starting body weight and ending body weight and expressed as g/kg bw/day

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations: every Monday, Tuesday, Wednesday, thursday and Friday for control, 0.5 mg/ml, 3 mg/ml, 7.5 mg/ml and 15 mg/ml dose group animals, respectively. Water consumption was measured gravimetrically, per animal, for 13 weekly intervals, and reported as g/animal/day.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 4 and 21 days of exposure in clinical pathology group, at 7 days of exposure from cell replication group, and at the end of the study (13 weeks) from core subgroup
The following endpoints were examined: red blood cell couint, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobine concentration, whte blood cell count, platelet count, reticulocyte count, white blood cell differential, morphological assessment

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 4 and 21 days of exposure in clinical pathology group, at 7 days of exposure from cell replication group, and at the end of the study (13 weeks) from core subgroup
- How many animals: 20 per dose level
Furthermore, trunk blood was collected, following decapitation, from animals of the clinical pathology subgroup at 28 days of exposure for hormone measurements in blood.

URINALYSIS: Yes
- Time schedule for collection of urine: after 4 and 21 days of exposure from male rats of clinical pathology group
- Metabolism cages used for collection of urine: Yes. n=4.
- Animals fasted: No
- Urine was examined for the following endpoints: appearance and color, osmolality, specific gravity, bilirubin (semi-quantitative), blood (semi-quantitative), glucose (semi-quantitative), ketones (semi-quantitative), pH, protein (semi-quantitative), urobilinogen (semi-quantitative), microscopic examination for casts, cells and crystals.

OTHER:
Approximately 3.5 days prior to euthanasia at 1, 4 and 13 weeks, rats included in the cell replication subgroup were implanted with 2ML1 osmotic pumps containing bromodeoxyuridine (BrdU). Kidneys were collected from male and females and the testes from males and wet weights were measured. The right kidney was frozen for analysis of α2u‐globulin and the left kidney, along with a section of duodenum, was immersed in first neutral‐buffered formalin (NBF) then ethanol for histopathology and BrdU immunohistochemistry. The left testis was immersed in modified Davidson’s fixative for histopathology.. The tissues were paraffin embedded, sectioned and immunostained for the presence of BrdU. Cell replication in the kidney was determined by counting BrdU stained proximal tubule cells in the cortex of the kidney. A minimum of 2000 cells were counted for each animal.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes control and high dose group only: Cardiovascular, respiratory, digestive, glandular, nervous, urogenital and skeletomuscular tissues were collected and selected tissues were weighed. Kidney, thyroid and eyes from all dose groups. Also testes, kidneys from cell replication subgroup (H&E stained). Staining for α2u-globulin from animals from 1, 4 and 13 weeks exposure groups.
Other examinations:
Cell replication in kidney and testes (cell replication subgroup). Frozen right kidneys from cell replication animals were thawed, weighed, diced and homogenized in 3 vols of PBS, centrifuged then supernatant assayed for D‐limonene.
Statistics:
Data meeting the assumption of normality and homogeneity of variance were analyzed using one-way ANOVA (p<0.05). Comparisons of means against control were performed using Dunnett's test (p<0.05) except in the case of urinalysis data where Duncan's test was used. Where the data did not meet the assumptions of normality and homegeneity it was analyzed using nonparametric tests (e.g. Kruskal-Wallis). The statistical packages JMP 6.0 (SAS, CAry, NC) or SAS (6.2 for Windows) were utilized to carry out the analyses.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
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):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
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:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no treatment-related deaths or clinical observations

BODY WEIGHT AND WEIGHT GAIN
Females: no significant effects. Males: there were significant differences between control and exposed males at weeks 12 and 13 for the 15mg/ml group and at week 13 for the 7.5mg/ml group.

FOOD CONSUMPTION
Food consumption was unaffected in either sex

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Effects seen at all dose levels with reductions compared to controls of 14, 23, 30, 35% in males and 22, 33, 40, 43% in females respectively at the four dose levels. There was also a decline in intake with time. Remark of the reveiwer: the decreased water consumption is likely due to poor palability of the drinking water containing MTBE and is therefore not taken into consideration as an adverse toxicological effect.

HAEMATOLOGY
No treatment-related changes were observed.

CLINICAL CHEMISTRY
Increased serum sodium levels were increased in males and females of the high dose group (likely to be increased due to reduced consumption of water).

URINALYSIS
Urine collected from male rats after 4 and 231 days of exposure demonstrated concentration of the urine, as increased specific gravity and osmolality, indicating an adjustment to decreased water intake.

ORGAN WEIGHTS
Males: Relative and absolute combined kidney weights were elevated compared to concurrent controls in the 7.5mg/ml dose group (Absolute: +7.6%; relative: +21%) and the 15mg/ml dose group (Absolute: +5.7%; relative: +19%).
Females: Relative combined kidney weights only were elevated compared to concurrent controls in the 7.5mg/ml dose group only (+21%). Relative and absolute combined kidney weights were elevated compared to concurrent controls in the 15mg/ml dose group (Absolute: +16%; relative: +30%)

GROSS PATHOLOGY
No treatment-related changes were observed.

HISTOPATHOLOGY: NON-NEOPLASTIC
Cell replication in cortical epithelial cells was increased at four weeks of exposure in males of the high dose group. Tubular cell regeneration was noted in males at one week in cell replication subgroup males (2/5, 1/5 and 1/5 for 0.5, 3, 15 mg/ml dose groups) and at 13 weeks in core subgroup animals (2/10, 3/10, 3/10 and 6/10 for 0.5, 3, 7.5 and 15 mg/ml dose groups, respectively). Alpha2µ-globulin levels, measured by an ELISA, in kidney were elevated in male, but not in female, rats after one and four weeks of exposure. Similarly, subjective scoring of male kidney sections stained for alpha2µ-globulin resulted in apparent increases in hyaline droplets after 1 and 13 weeks of exposure.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
3 000 mg/L drinking water
Sex:
male
Basis for effect level:
other: Body weight, kidney effects (weight, cell replication and α2u-globulin levels, tubular cell regeneration). Dose equivalent to 209mg/kg/day
Dose descriptor:
NOAEL
Effect level:
3 000 mg/L drinking water
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Relative combined kidney weight. Dose equivalent to 272mg/kg/day

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

MTBE air concentrations averaged 15ppm for females and 28ppm for males in the high dose cages (maximum values 158 and 181ppm respectively). Control cages averaged 0.34ppm.

Tertiary butyl alcohol was measured in blood taken at terminal necropsy and found to significantly correlate with exposure concentration of MTBE.

Applicant's summary and conclusion