2-ethoxy-2-methylpropane

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

chronic toxicity: inhalation

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, 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 exposed to the test substance via inhalation (whole body exposure) at 0, 500, 1500 and 5000 ppm (v/v), 6 hr/d, 5 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 114-137 g, females 90-104 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 (%): 40-70%
- Air changes (per hr): 15-17 per hr
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 22 May 2007 (initiation of treatment) To: 19-25 May 2009 (scheduled necropsy)

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

clean air

Remarks on MMAD:

MMAD / GSD: vapour exposure, not applicable

Details on inhalation exposure:

Animals were exposed 6 hr/d, 5 d/wk for 104 weeks (520 exposures in total).

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: multiple, rectangular-pyramid stainless steel chambers (volume 1.11 m3, Sibata Scientific Technology Ltd)
- Method of holding animals in test chamber: individually housed in stainless steel wire-mesh cages
- System of generating atmosphere: warmed compressed air passed through reservoir of test substance, then mixed with more compressed air for secondary dilution
- Air change rate: 13 /hr
- Mean temperature in chamber: 22 degrees C
- Mean humidity in chamber: 53-57%
- Treatment of exhaust air: activated charcoal/water washing prior to release to atmosphere

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber concentrations were monitored by GC-FID (Agilent 5890A with Shimadzu GC-14B automatic sampler) every 15 min, and the supply into the inhalation chamber regulated to maintain the target concentration. Results of these analyses showed that differences between the actual and intended concentrations were less than 0.1 %.

Mean overall analyzed concentrations were 500.5 (+/-4.7), 1501.3 (+/- 12.5) and 4998.8 (+/- 47.5) ppm.

Duration of treatment / exposure:

104 weeks

Frequency of treatment:

6 hr/d, 5 d/wk

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 500, 1500 and 5000 ppm (v/v)
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 500.5, 1501.3 and 4998.8 ppm (v/v)
Basis:
analytical conc.

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 exposed to 0 (air), 50, 150, 500, 1500 and 5000 ppm (v/v) ETBE vapour (6 hr/d, 5 d/wk 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 marked reduction in body weight was noted primarily in high dose males (-11%; -5% reduction in females). Haematological assessment indicated slight but significant decreases in red blood cell count and haemoglobin concentration, and slight but significant increases in platelet count, in 5000 ppm group males which also exhibited statistically significant alterations in blood protein, lipids, and urea nitrogen. Kidney weights were significantly increased in both sexes at 1500 ppm and 5000 ppm ETBE, with significantly increased liver weights in both sexes at 5000 ppm. Microscopic assessment revealed an increased incidence of regeneration of proximal renal tubules in 5000 ppm males. An upper treatment level of 5000 ppm (v/v) was therefore set for the main study, with intermediate and low dose levels set at 1500 and 500 ppm (proportional factor of 3).

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

- Vapour generation: ETBE was placed in an organic solvent vapor generator (Shibata Scientific Technology Ltd, Tokyo, Japan), and vaporized by bubbling with warmed clean air. The resulting saturated vapor was diluted with warmed clean air and introduced into the exposure chambers via a flow meter.

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.

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):

no effects observed

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:

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:
Survival was decreased significantly in the intermediate (females only) and high (both sexes) treatment groups:
Male survival at week 104: 88%, 76%, 80% and 60% for the 0, 500, 1500 and 5000 ppm groups, respectively.
Female survival at week 104: 76%, 78%, 60% and 60% for the 0, 500, 1500 and 5000 ppm groups, respectively.

BODY WEIGHT:
Males - decreased significantly at 5000 ppm throughout the course of the test, with reductions in the low- and intermediate groups towards the end of the exposure period. Body weights at week 104 in the 0, 500, 1500 and 5000 ppm groups were 426 (100%), 402 (94% ; P<0.01), 402 (94%; P<0.01) and 321 (75%; P<0.01)
Females – decreased significantly at 5000 ppm from week 10 until termination and in the 1500 ppm group from week 30 until study end. Body weights at week 104 in the 0, 500, 1500 and 5000 ppm groups were 276 g (100%), 262 g (95%; n/s), 251 g (91%; P<0.01) and 214 g (78%; P<0.01), respectively.

HAEMATOLOGY
Males – no biologically relevant changes present
Females – very slight, statistically significantly reductions in MCV (-3%) and MCH (-2%) in the 5000 ppm group.

CLINICAL CHEMISTRY:
Blood urea nitrogen, total cholesterol, phospholipid and potassium were statistically significantly increased, and A/G ratio, albumin and AST significantly decreased, at one or more treatment levels in both sexes.
- Males -
Urea nitrogen, creatinine, and inorganic phosphorus were increased significantly in all treated groups, with total cholesterol, triglyceride, phospholipid, gamma-GTP and calcium significantly increased, and A/G ratio significantly decreased, at 1500 and 5000 ppm. Potassium was significantly increased, and total protein, albumin, AST and chlorine significantly decreased, at 5000 ppm.
- Females -
Total cholesterol, phospholipid, urea nitrogen and potassium were significantly increased, and albumin, A/G ratio, AST, ALT and sodium significantly decreased, in the 5000 ppm group.

URINE ANALYSIS:
Males – pH decreased significantly in all treated groups.
Females – pH significantly decreased, and protein significantly increased, at 5000 ppm.

GROSS NECROPSY:
Males –
kidney: dose-related increase in occurrence of granulated surface of the kidney (affecting 9, 16, 17 and 36 animals from the 0, 500, 1500 and 5000 ppm groups, respectively).
Females -
Kidney: increased occurrence of granulated surface of the kidney at 5000 ppm (affecting 2, 1, 0 and 6 animals from the 0, 500, 1500 and 5000 ppm groups, respectively).

ORGAN WEIGHTS:
Males -
kidney: statistically significant increases in absolute and relative weights in all treated animals
- absolute kidney weight (g): 2.18, 3.03 (P<0.05), 3.28 (P<0.01) and 3.44 (P<0.01) for the 0, 500, 1500 and 5000 ppm groups, respectively.
- relative kidney weight (g/100 g bw): 0.71, 0.85 (P<0.01), 0.90 (P<0.01) and 1.18 (P<0.01) for the 0, 500, 1500 and 5000 ppm groups, respectively.
liver: significantly elevated relative weight in all treated groups
- relative liver weight (g/100 g bw): 3.01, 3.29 (P<0.01), 3.57 (P<0.01) and 4.47 (P<0.01) for the 0, 500, 1500 and 5000 ppm groups, respectively. (Note: no histopathological alterations present in low or intermediate treatment groups.)
In addition, relative heart, lung and brain weights were also significantly increased in all treated groups, with relative spleen weight increased at 1500 and 5000 ppm. Relative adrenal and testis weights were increased significantly at 5000 ppm only

Females -
kidney: statistically significant increased absolute weight present at 1500 and 5000 ppm, with relative weights increased significantly in all treated animals
- absolute kidney weight: 1.81, 1.90, 1.92 (P<0.05) and 2.13 (P<0.01) for the 0, 500, 1500 and 5000 ppm groups, respectively.
- relative kidney weight (g/100 g bw): 0.71, 0.80 (P<0.01), 0.83 (P<0.01) and 1.08 (P<0.01) for the 0, 500, 1500 and 5000 ppm groups, respectively.
liver: significantly elevated relative weight in intermediate and high treatment groups
- relative liver weight (g/100 g bw): 2.75, 2.83, 2.79 (P<0.01) and 3.58 (P<0.01) for the 0, 500, 1500 and 5000 ppm groups, respectively. (Note: no histopathological alterations present in any treatment group.)
In addition, relative heart and brain weights were also significantly increased in all treated groups, with relative ovary and lung weights increased at 1500 and 5000 ppm. Relative adrenal weight was increased significantly at 5000 ppm only.

HISTOPATHOLOGY – NON-NEOPLASTIC LESIONS
Males -
Liver: the incidence of acidophilic and basophilic cell foci (pre-neoplastic lesions) was increased significantly in the 5000 ppm group:
- acidophilic cell foci: 31/50 (slight), 28/50 (slight), 36/49 (slight) and 39/50 (30 slight, 9 moderate) for the 0, 500, 1500 and 5000 ppm groups, respectively
- basophilic cell foci: 18/50 (slight), 10/50 (slight), 13/49 (slight) and 33/50 (31 slight, 2 moderate) for the 0, 500, 1500 and 5000 ppm groups, respectively
The incidence of bile duct hyperplasia was significantly decreased at 5000 ppm.

Kidney: 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) and mineral deposition in the renal papilla at 5000 ppm, with significantly increased incidences of urothelial hyperplasia of the pelvis (characterised by multilayer thickening of the papillary epithelium with protrusion into the pelvi) at 1500 and 5000 ppm.

Stomach:
Mineral deposition was found in 1/50 (slight), 3/50 (1 slight, 2 moderate), 0/49 and 9 /50 (6 slight, 3 moderate) rats from the 0, 500, 1500 and 5000 ppm groups, respectively.

Artery/aorta:
Mineral deposition in arteries was found in 2 /50 rats in the 500 ppm group (1 slight, 1 severe) and 6 /50 rats in the 5000 ppm group (slight), with a significantly increased incidence in the 5000 ppm group.

Females -
Kidney: significant increase in the degree of chronic nephropathy present at 5000 ppm.
(The extent of mineral deposition in the renal papilla and incidences of urothelial hyperplasia of the pelvis were comparable between the control and treated groups).

Effect levels

1

Dose descriptor:

LOEC

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: approx. 5% reduction in terminal body weight, considered of doubtful toxicological relevance.

2

Dose descriptor:

LOEC

Effect level:

500 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

3

Dose descriptor:

NOAEC

Effect level:

500 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: altered clinical chemistry parameters (total cholesterol, triglyceride, phospholipid, gamma-GTP, calcium, A/G ratio) at 1500 ppm.

4

Dose descriptor:

NOAEC

Effect level:

500 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: decreased survival at 1500 and 5000 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	1	24	23	1
500	50/50	2	20	24	4
1500	49/49	0	17	31	1
5000	50/50	0	12	19	19

 

Urothelial hyperplasia:

Group (ppm)	Number affected	Grade
		slight	moderate	marked	severe
0	2/50	2	0	0	0
500	5/50	5	0	0	0
1500	16/49	16	0	0	0
5000	41/50	41	0	0	0

 

Mineral deposition in renal papilla:

Group (ppm)	Number affected	Grade
		slight	moderate	marked	severe
0	0/50	0	0	0	0
500	0/50	0	0	0	0
1500	1/49	1	0	0	0
5000	6/50	6	0	0	0

 

HISTOPATHOLOGY – NON-NEOPLASTIC LESIONS - FEMALES

Nephropathy:

Group (ppm)	Number affected	Grade
		slight	moderate	marked	severe
0	32/50	19	11	2	0
500	38/50	13	24	1	0
1500	41/50	19	19	3	0
5000	40/50	10	20	9	1