2-ethoxy-2-methylpropane

Repeated dose toxicity: oral

Administrative data

Endpoint:

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 near-guideline study, no restrictions, fully adequate for assessment

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Principles of method if other than guideline:

The potential chronic toxicity and carcinogenicity of ETBE was investigated in male and female rats given drinking water containing the test substance at 0, 625, 2500 or 10000 ppm, 7 d/wk for 104 weeks. Animals were examined for general condition, body weight, food consumption, water consumption, haematology, clinical chemistry, urinalysis, gross pathology, organ weights and histopathology.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-ethoxy-2-methylpropane

EC Number:

211-309-7

EC Name:

2-ethoxy-2-methylpropane

Cas Number:

637-92-3

Molecular formula:

C6H14O

IUPAC Name:

2-ethoxy-2-methylpropane

Details on test material:

ETBE, lot # l-506251, greater than 99 wt% pure (analysis by Toray Research Center Co. Ltd, Tokyo, Japan) was supplied by Nippon Refine Co. Ltd, Gifu, Japan. The identity of the test substance was verified by electron impact mass spectrometry.

Test animals

Species:

rat

Strain:

Fischer 344/DuCrj

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan Inc., Kanagawa, Japan
- Age at study initiation: 6 wk
- Weight at study initiation: males 121-142g, females 96-111 g
- Housing: individually housed in stainless steel mesh cages
- Diet (ad libitum): CRF-1 (irridated pellets, Oriental Yeast Co. Ltd, Chiba, Japan)
- Water (ad libitum): deionized tap water (filtered, UV iradiated, deionized, re-filtered)
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25 degrees
- Humidity (%): 50-60%
- Air changes (per hr): 15-17 per hr
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 20 June 2007 (initiation of treatment) To: 18-24 June 2009 (scheduled necropsy)

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- Concentration in vehicle: 0 (sterile deionized water), 625, 2500 or 10000 ppm (w/w)
- Frequency of preparation: weekly

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Achieved concentration: test solutions were analyzed using GC-FID (Agilent Technologies 5890A with head-space sampler). Results were within 10% of target.

Stability: test solutions containing 250 or 10000 ppm ETBE were stored at room temperature for 8 days, then analyzed as above. Results were within 10% of target.

Duration of treatment / exposure:

104 weeks

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 625, 2500 or 10000 ppm (w/w)
Basis:
nominal in water

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 28, 121 or 542 mg/kg bw/d (males)
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
0, 46, 171 or 560 mg/kg bw/d (females)
Basis:
actual ingested

No. of animals per sex per dose:

50

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: treatment levels were based upon a preliminary study in which male and female F344/DuCrlCrlj rats were given free access to drinking water containing 0 (control), 250, 640, 1600, 4000 or 10000 ppm (w/w) ETBE for 13 weeks. Detailed information from this investigation is unavailable for independent review, however key findings are discussed briefly in the report of this 2-year study. There was no mortality, however a significant reduction in body weight and a significant increase in kidney weight was apparent in high-dose animals of both sexes, with renal histopathology present in males. Slight but statistially significant reductions in red cell count and haemoglobin concentration were noted in females exposed to 640 ppm and above, with slight but significant increases in urea nitrogen in males over the same treatment range. An upper treatment level of 10000 ppm was therefore set for the main study, with intermediate and low dose levels set at 2500 and 625 ppm (proportional factor of 4). The report notes that the high-dose concentration of 10000 ppm (w/w) is close to the solubility limit of ETBE in water (12000 ppm w/w).
- Drinking water was supplied from a pressurised tank (0.05 mPa) to each cage, and the animals allowed to drink ad libitum. The contents of the tank were replaced once each week.

- Rationale for animal assignment (if not random): stratified according to body weight

Positive control:

- not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
Checked daily for general condition.

DETAILED CLINICAL OBSERVATIONS:
Subject to a weekly detailed examination.

BODY WEIGHT:
Animals weighed weekly until week 14, then once every 4 weeks thereafter. Terminal body weights recorded at week 104 (or at time of death for decedent animals).

FOOD CONSUMPTION:
Food intake determined weekly until week 14, then once every 4 weeks thereafter (plus additionally at week 104). Food consumption per animal calculated.

WATER CONSUMPTION AND COMPOUND INTAKE:
Water intake determined weekly until week 14, then once every 4 weeks thereafter (plus additionally at week 104). Water consumption per animal calculated together with received dose of test substance.

HAEMATOLOGY:
Haematological examinations were performed on surviving animals at study termination. Blood was collected from the abdominal aorta under ether anesthesia, just before necropsy, into sample tubes coated with EDTA-2 potassium. The following parameters were examined: red cell count, haemoglobin concentration, haematocrit, mean cell volume (MCV), mean corpuscular haemoglobin (MCH), mean cell haemoglobin concentration (MCHC), platelet count, reticulocyte count, white cell count, differential white cell counts and percentages.

CLINICAL CHEMISTRY:
Clinical chemistry examinations were performed on surviving animals at study termination. Blood was collected from the abdominal aorta under ether anesthesia, just before necropsy, into sample tubes coated with heparin lithium. The following parameters were examined: total protein, albumin, A/G ratio, total bilirubin, glucose, total cholesterol, triglyceride, phospholipid, AST, ALT, LDH, ALP, y-GTP, CK, urea nitrogen, creatinine, sodium, potassium, chlorine, calcium, inorganic phosphorus.

URINALYSIS:
Fresh urine was obtained from animals that survived to week 104, and the following parameters analyzed using urine test paper (Multisticks, Siemens Healthcare Diagnostics Inc.): pH, protein, glucose, ketones, bilirubin, occult blood, urobilinogen.

Sacrifice and pathology:

GROSS PATHOLOGY:
All animals were subject to a full gross necropsy.

ORGAN WEIGHTS:
The following organs were weighed at scheduled necropsy: adrenal, testis, ovary, heart, lung, kidney, spleen, liver, and brain. Relative organ weights were calculated (based on organ:body weight ratios).

HISTOPATHOLOGY:
The following tissues were sampled from all treatment groups at scheduled termination, fixed (10% neutral buffered formalin solution), embedded (paraffin wax blocks) and stained with haematoxylin and eosin: skin, nasal cavity (at 3 levels), nasopharynx, larynx, trachea, lung, bone marrow (femur), lymph node (axilla, abdominal wall), thymus, spleen, heart, tongue, salivary glands, oesophagus, stomach, small intestine (including duodenum), large intestine, liver, pancreas, kidney, urinary bladder, pituitary gland, thyroid gland, parathyroid, adrenal gland, testis, epididymis, seminal vesicle, prostate, ovary, uterus, vagina, mammary gland, brain, spinal cord, peripheral nerve (sciatic nerve), eyeball, Hardrian gland, muscle, bone (femur), and any other tissues with an abnormal appearance macroscopically. Lesions were graded as 0 (no lesion present) or grade 1-4 dependent on the extent of the alteration present.

Statistics:

Statistical comparisons of body weights, food consumption, haematology, clinical chemistry and organ weights were performed using Bartlett's test followed by ANOVA and Dunnett’s test if the data were homogenous data; or the Kruskal-Wallis test followed by Dunnett’s multiple comparison test for non-homogeneous results. Histopathological (non-tumour) data were analysed using the Chi-square test.

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):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

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:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Details on results:

SURVIVAL:
There were no effects on survival in either sex:
Male survival at week 104: 76%, 74%, 68%, 68% for the 0, 625, 2500 and 10000 ppm groups, respectively;
Female survival at week 104: 72%, 74%, 76%, 76% for the 0, 625, 2500 and 10000 ppm groups, respectively.

BODY WEIGHT:
Males - decreased significantly at 2500 ppm or 10000 ppm throughout the course of the test, and decreased significantly in the 625 ppm group from week 26. Body weights at week 104 in the 0, 625, 2500 and 10000 ppm groups were 400 g (100%), 382 g (96%; n/s), 372 g (93%; P<0.01) and 363 g (91%; P<0.01), respectively.
Females – decreased significantly at 10000 ppm throughout the study, and from week 38 and week 14 for the 625 ppm and 2500 ppm groups, respectively. Body weights at week 104 in the 0, 625, 2500 and 10000 ppm groups were 290 g (100%), 261 g (90%; P<0.01), 259 g (89%; P<0.01) and 241 g (83%; P<0.01), respectively.

FOOD CONSUMPTION:
Males - average food consumption in the 0, 625, 2500 and 10000 ppm groups was 15.4 g (100%), 14.9 g (97%), 14.9 g (97%) and 14.8 g (96%), respectively.
Females - average food consumption in the 0, 625, 2500 and 10000 ppm groups was 10.8 g (100%), 10.3 g (95%), 10.5 g (97%) and 10.3 g (95%), respectively.

WATER CONSUMPTION:
Males – significantly decreased water consumption was found in the 625 and 2500 ppm groups throughout the treatment period, and in the 10000 ppm group during the early phase (before week 22). Average water consumption throughout the study in the 0, 625, 2500 and 10000 ppm groups was 18.1 g (100%), 14.6 g (81%), 15.4 g (85%), and 17.1 g (94%), respectively.
Females - significantly decreased water consumption was found in all treated groups throughout the treatment period. Average water consumption throughout the study in the 0, 625, 2500 and 10000 ppm groups was 17.8 g (100%), 13.7 g (77%), 13.0 g (73%), and 10.3 g (58%), respectively.

INTAKE OF ETBE:
Males - test substance intake for the 625, 2500 and 10000 ppm groups was in a range 21-59, 92-236 and 439-792 mg/kg/day, respectively. Respective average values were 28, 121 and 542 mg/kg/day
Females - test material intake in the 625, 2500 and 10000 ppm groups was in a range 31-74, 134-267 and 460-903 mg/kg/day, respectively. Respective average values were 46, 171 and 560 mg/kg/day.

HAEMATOLOGY
Males – no changes present
Females – significantly decreased MCV at 625 (-7%) and 10000 ppm (-7%).

CLINICAL CHEMISTRY:
Males - urea nitrogen was increased significantly in the 2500 or 10000 ppm groups. Total cholesterol, triglyceride, phospholipid, and inorganic phosphorus were significantly increased, and total protein and albumin significantly decreased, in the 10000 ppm group.
Females - total bilirubin, AST, LDH and chlorine were decreased significantly in the 10000 ppm group.

URINE ANALYSIS:
Males – pH decreased significantly in the 10000 ppm group.
Females – incidence of animals testing positive for occult blood increased significantly in the 10000 ppm group.

GROSS NECROPSY:
Males –
kidney: increased occurrence of granulated surface of the kidney at 10000 ppm group (affecting 4, 2, 5 and 9 animals from the 0, 625, 2500 and 10000 ppm groups, respectively).
spleen: tendency for enlarged spleen in treated animals (affecting 3, 5, 7 and 7 animals form the 0, 625, 2500 and 10000 ppm, respectively). Enlarged spleens commonly accompanied by leukemia, but no statistically significant increase in incidence present.
Females-
thyroid: increased occurrence of enlarged thyroids at 10000 ppm (affecting 1, 0, 2 and 5 animals from the 0, 625, 2500 and 10000 ppm groups, respectively). Animals with enlarged thyroid had thyroid tumours (C-cell adenoma or C-cell carcinoma), but no statistically significant increase in tumour incidence present.

ORGAN WEIGHTS:
Males -
kidney: statistically significant increase in absolute weights at 10000 ppm (+18%), significant increase in relative weights at 2500 (+13%) and 10000 ppm (+32%).
liver: generally non-significant reductions in absolute weight in all treated groups, significantly elevated relative weight at 2500 (+3%) and 10000 ppm (+12%).
In addition, relative lung and brain weights were significantly increased at 2500 and 10000 ppm, with relative adrenal, heart and spleen weights increased significantly at 10000 ppm only.
Females -
kidney: statistically significant increase in absolute weights at 2500 (+10%) and 10000 ppm (+14%), significant increase in relative weights in all treated groups (+14%, +23% and +37%).
liver: no obvious treatment effect on absolute or relative weights.
In addition, relative heart, lung and brain weights were significantly increased in all treatment groups, with relative adrenal weight increased at 2500 and 10000 ppm.

HISTOPATHOLOGY – NON-NEOPLASTIC LESIONS
Males -
Significant increase in the degree of chronic nephropathy (comprising sclerosis of glomeruli, thickening of the renal tubular basement membranes, hyaline casts in renal tubules, regeneration of renal tubules, interstitial fibrosis and inflammatory cell infiltration) at 10000 ppm, together with significantly increased incidences of urothelial hyperplasia of the pelvis (characterised by multilayer thickening of the papillary epithelium with protrusion into the pelvi) and mineral deposition in the renal papilla at 2500 and 10000 ppm.
No changes in microscopic appearance of liver tissue (despite increased relative weights).
Microscopic assessment of other tissues revealed no other treatment-related changes.

Females -
A significant increase in the degree of chronic nephropathy was observed in the 10000 ppm group.
Microscopic assessment of other tissues revealed no other treatment-related changes.

Effect levels

1

Dose descriptor:

LOEL

Effect level:

625 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOEL

Effect level:

625 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

3

Dose descriptor:

NOAEL

Effect level:

2 500 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: (equiv. to approx. 121 mg/kg bw/d): altered clinical chemistry parameters (total cholesterol, triglyceride, phospholipid, inorganic phosphorus, total protein, albumin) at 10000 ppm.

4

Dose descriptor:

NOAEL

Effect level:

2 500 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: (equiv. to approx. 171 mg/kg bw/d): altered clinical chemistry parameters (total bilirubin, AST, LDH, chlorine) at 10000 ppm.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

HISTOPATHOLOGY – NON-NEOPLASTIC LESIONS - MALES

Nephropathy:

Group (ppm)	Number affected	Grade
		slight	moderate	marked	severe
0	49/50	4	37	7	1
625	43/50	5	32	6	0
2500	45/50	6	32	7	0
10000	48/50	6	21	19	2

 

Urothelial hyperplasia:

Group (ppm)	Number affected	Grade
		slight	moderate	marked	severe
0	0/50	0	0	0	0
625	0/50	0	0	0	0
2500	10/50	10	0	0	0
10000	25/50	25	0	0	0

 

Mineral deposition in renal papilla:

Group (ppm)	Number affected	Grade
		slight	moderate	marked	severe
0	0/50	0	0	0	0
625	0/50	0	0	0	0
2500	16/50	16	0	0	0
10000	42/50	25	17	0	0

 

 

HISTOPATHOLOGY – NON-NEOPLASTIC LESIONS - FEMALES

Nephropathy:

Group (ppm)	Number affected	Grade
		slight	moderate	marked	severe
0	41/50	31	10	0	0
625	37/50	29	7	1	0
2500	37/50	22	13	2	0
10000	39/50	26	5	8	0