3-ethyloxetane-3-methanol

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25th November 1996 - 30th July 1997.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Principles of method if other than guideline:

Not relevant.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-ethyloxetane-3-methanol

EC Number:

221-254-0

EC Name:

3-ethyloxetane-3-methanol

Cas Number:

3047-32-3

Molecular formula:

C6H12O2

IUPAC Name:

(3-ethyloxetan-3-yl)methanol

Specific details on test material used for the study:

- Name of test material (as cited in study report): Oxetane (OH)
- Molecular formula (if other than submission substance): C6H12O2
- Molecular weight (if other than submission substance): 116.16
- Physical state: Clear and colourless liquid.
- Analytical purity: More than 99.9%
- Lot/batch No.: 960501-4
- Stability under test conditions: Stable during the dosing period.
- Storage condition of test material: Store in a cold and dark place.
- Other: Melting point: -37°C
Boiling point: 105°C/7mmHg
Solubility: Easily soluble in water and soluble in acetone, methanol and toluene.

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc, Hino Breeding Centre, 735, Shimokomatsuki, Hino-cho, Gamo-gun, Shiga 529-16, Japan.
- Age at study initiation: 5 weeks old.
- Weight at study initiation: Males - 121.2 - 136.8g and females - 105.6 - 120.8g
- Fasting period before study: No fasting before study period.
- Housing: Housed individually in a hanging stainless steel cage with wire-mesh floor (165W x 300D x 150H mm) in a barrier system animal room during the study periods.
- Diet (e.g. ad libitum): Free access to an MF pelleted diet (Oriental Yeast)
- Water (e.g. ad libitum): Free access to chlorinated drinking water that was provided from the Hita City supply via automatic watering system with sipper tubes.
- Acclimation period: Animals were acclimatized.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2°C
- Humidity (%): 55 ± 10%
- Air changes (per hr): 10 - 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark.

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was weighed accurately and dissolved in purified water with agitation to obtain 10 w/v % solution. Lower concentrations of 2 and 0.4 w/v% were prepared from a 10 w/v% solution by dilution with purified water. Preparations were performed once a week.

The test substance was administered using a Nelaton cathether (Terumo) and a syringe (Terumo) in the morning for 28 days.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No information provided.

Duration of treatment / exposure:

28 days.

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Vehicle control

Doses / concentrations 2

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

6 males and 6 females per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for Dosage Selection:
A preliminary repeated-dose oral toxicity study was carried out over 14 days at concentrations of 0, 50, 250 and 1000 mg/kg. Due to the abnormal results observed in clinical signs, blood chemical examinations and organ weights at the highest dose level, the maximum dose level in the 28 day study was determined to be 1000 mg/kg and the two lower doses selected were 200 and 40 mg/kg.

Positive control:

No information provided.

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed at least once per day.

BODY WEIGHT: Yes
- Time schedule for examinations: Test animals were weighed the day before dosing, during the dosing period on days 1, 3, 5, 8, 10, 12, 15, 17, 19, 22, 24, 26 and 28 and during the recovery period days 1, 3, 5, 8, 10, 12 and 14.
Body weights were also measured immediately before necropsy for calculation of relative organ weights.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption was measured once before dosing and twice a week during the dosing and recovery periods.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were taken at the termination of the dosing and recovery periods.
- Anaesthetic used for blood collection: Yes - ether anaesthesia was used.
- Animals fasted: Yes for 16 - 20 hours.
- How many animals: All surviving animals.
- Parameters examined included Red bloodcell count, white blood cell count, haemoglobin concentration, haematocrit calue, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelet concentration, reticulocytes count, phrothrombin time, activated partial thromboplastin time and differentiation of leukocytes.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were taken at the termination of the dosing and recovery periods.
- Animals fasted: Yes
- How many animals: All surviving animals.
- Parameters examined included GOT, GPT, alkaline phosphatase, cholinesterase, ϒ-GTP, total cholesterol, triglyceride, glucose, total protein, albumin, A/G ratio, blood urea nitrogen, creatinine, total bilirubin, calcium, inorganic phosphorous, sodium, potassium, chloride.

URINALYSIS: Yes
- Time schedule for collection of urine: 16 hour urine samples from all animals at day 28 and recovery day 14
- Metabolism cages used for collection of urine: Yes - collected in individual metabolic cages
- Animals fasted: No data
- Parameters examined: Volume, colour, pH, protein, ketone bodies, bilirubin, occult blood, glucose and urobilinogen.

OTHER:
Organ Weights: The brain, liver, kidneys, spleen, adrenals and testes/ovaries were weighed wet in all animals.

HISTOPATHOLOGICAL EXAMINATIONS
The following organs and tissues from all animals were preserved and fixed in 10% formalin for histopathological examinations:
Brain (cerebrum, cerebellum), pituitary gland, eyeball, thyroid (with parathyroid), heart, lung, liver, kidneys, spleen, adrenals, stomach, intestine (duodenum to rectum), testes (or ovaries), urinary bladder, bone marrow (femur) and macroscopic lesions.

Sacrifice and pathology:

Necropsy:
All animals were subjected to a detailed gross necropsy.

Histopathological examinations:
The brain (cerebellum and cerebrum), pituitary gland, eyeball, thyroid (with parathyroid), heart, lung, liver, kidneys, spleen, adrenals, stomach, intestine (duodenum to restum), testes (or ovaries, urinary bladder, bone marrow (femur) and macroscopic lesions were perserved and fixed in 10% formalin.
Light microscopic examinations were performed on the following organs and tissues after paraffin embedding and sectioning followed by haemotoxylin-eosin staining.
At terminal necropsy of the dosing period, in the vehicle control and the 1000 mg/kg dosing group, the liver, kidneys, spleen, heart, stomach, intestine (duodenum, jejunum, cecum, colon and rectum ) and the adrenals were examined. At terminal necropsy of the recovery period, in the vehicle control and the 1000 mg/kg dosing group only the livers from the male test animals were examined.
Macroscopic lesions were examined at terminal necropsy of the dosing period. In the 40 mg/kg dose grouop, the skin and submandibular lymph node were examined in one male and the glandular stomach was examined in one female. In the 200 mg/kg dose grou, the cerebrum was examined in one female.

Other examinations:

No other examinations were conducted.

Statistics:

Data relating to body weights, food intakes, haematological examinations, blood chemical examinations, urine volume and organ weights were analysed using Bartlett's test for homogeneity of variance. If the variances were homogeneous at a significance level of 5%, one way analysis of variance was performed. When there was a significant difference in this analysis, the difference between the vehicle control group and each of the treatment groups was analysed by Dunnett's test.
If the variances were not homogeneous in the Bartlett's test, Kruskal-Wallis's test was used. When there was a significant difference in this test, the difference between the vehicle control group and each of the treatment groups was analyzed by nonparametric Dunett's test.

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:

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

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
During Dosing Period:
There were no deaths during the dosing period.
Salivation was observed in all males in the 1000 mg/kg dose. The salivation was sporadically or continuosly observed just after dosing from day 5 or day 13. Hair loss (in one male) and exudates (in one male) were also observed in the 40 mg/kg dose group.
In females, salivation was observed in 10 out of 12 females in the 1000 mg/kg dose group. The salivation observed was sporadic or continuous from days 8 to 23 just after dosing.

No abnormalities were observed in either sex during the recovery period.

BODY WEIGHT AND WEIGHT GAIN
No abnormalities were observed in either sex during the dosing or recovery period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No abnormalities were observed in either sex during the dosing or recovery period.

HAEMATOLOGY
At termination of the dosing period, in males and females, a trend toward a decrease in differentiation of eosinophils was observed in the 1000 mg/kg dose group. In addition to this, in females, an elongated activated partial thromboplastin time was noted in the 40 mg/kg dose group.

At termination of the recovery period, in males, an increase in the mean corpuscular volume was noted in the 1000 mg/kg dose group. There were no abnormalities observed in the females of any dose group.

CLINICAL CHEMISTRY
At termination of the dosing period, in the 40 and 1000 mg/kg dose groups, a decrease in glucose was observed in males. A decrease in alkaline phosphatase was noted in females in the 40 mg/kg dose group.

At termination of the recovery period, in males dosed with 1000 mg/kg, a decrease in alkaline phosphatase, total bilirubin and sodium and increase in ϒ-GTP was observed. There were no abnormalities observed in the females of any dose group.

URINALYSIS
At termination of the dosing period, a trend towards an acidic pH was observed in the males and females in the 1000 mg/kg dose group. In addition to this, females in the 1000 mg/kg dose group showed a trend toward positive ketone bodies.

No abnormalities were observed in either sex during the recovery period.

ORGAN WEIGHTS
At termination of the dosing period, no abnormalities were observed in either sex during the recovery period.

At termination of the recovery period, in males treated with 1000 mg/kg, an increase in relative liver weight was observed. There were no abnormalities observed in the females of any dose group.

GROSS PATHOLOGY
At termination of the dosing period, in males treated with 40 mg/kg, one male displayed an enlargement of the submandibular lymph node, one male displayed exudation and one male showed loss of hair. In the 200 mg/kg dose group, one male had a wound on the hind limbs.
In females treated with 40 mg/kg, one female had a blackish region on the mucosa in the glandular stomach. In the 200 mg/kg dose group, dilatation of the ventricles in the cerebrum was observed in one female.

At termination of the recovery period, no abnormalities were observed in either sex at any dose level.

HISTOPATHOLOGY: NON-NEOPLASTIC
At the termination of the dosing period, in the 40 mg/kg treatment group, increased plasmacytes in the submandibular lymph node in one male and ulceration on the skin was also observed in one male. In the 1000 mg/kg treatment group, one male had basophilic tubules and one male had cell infiltration in the kidney. In the vehicle control group, one male had basophilic tubules and one male had cell infiltration in the kidney.
In females in the vehicle control group, one female displayed microgranuloma in the liver and one female each showed basophilic tubules, cell infiltration, cyst formation and fibrosis in the kidney. In the 40 mg/kg dose group, one female had necrosis of the mucosa in the glandular stomach. In the 200 mg/kg dose group, one female displayed dilatation of the ventricles in the cerebrum.

At termination of the recovery period, in males, no abnormalities were observed in any treatment group. Females were not examined at the end of the recovery period.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 other: mg/kg/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

No additional information.

Applicant's summary and conclusion

Conclusions:

The NOEL of oxetane (OH) for rats was determined to be 1000 mg/kg/day under the conditions of the study.

Executive summary:

A 28 day repeated-dose oral toxicity study was conducted on male and female Crj: CD (SD) rats using oxetane (OH). The test substance was administered by oral gavage once daily for 28 days at concentrations of 40, 200 and 1000 mg/kg. Six test animals per sex were used in each test group. Recovery groups were separately provided for the 1000 mg/kg and vehicle control groups. There were no mortalities observed throughout the course of the dosing period or the recovery period. In all the parameters examined, including clinical signs, body weight and food consumption during the dosing period, haematological and blood chemical parameters, urinalyses, organ weights, necropsy and histopathological examinations at the end of the dosing period, no abnormalities were observed which were attributed to administration of the test material. There were no abnormalities observed during the recovery period which were related to the test substance. In conclusion, the NOEL of oxetane (OH) for the rat was determined to be 1000 mg/kg/day under the conditions of the study.