3-ethyloxetane-3-methanol

Endpoint summary

Description of key information

28 -day and 90 -day studies of repeated dose toxicity study are available for the submission substance.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

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

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.

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:

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

Dose / conc.:

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

Remarks:

Vehicle control

Dose / conc.:

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

Dose / conc.:

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

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.

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.

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.

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

Key result

Critical effects observed:

no

No additional information.

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.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 March 2016 to 27 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Specific details on test material used for the study:

Identity TMPO
Alternative name Trimethylolpropane Oxetane
Batch no. 160100167
Purity 99.7%
Expiry date 13 January 2017
EC Name 3-ethyloxetane-3-methanol
EC No. 221-254-0
IUPAC Name (3-ethyloxetan-3-yl)methanol
CAS No. [3047-32-3]
Storage conditions Room temperature

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley SD rat was the species and strain of choice because it is accepted by many regulatory authorities and there is ample experience and background data on this species and strain.
The oral route was selected as it is a possible route of exposure of the test item in man.
Dose levels were selected based on information from preliminary studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS srl, San Pietro al Natisone (UD), Italy
- Age at study initiation: Seven to eight weeks
- Weight at study initiation: Male rats (220.4 to 248.9g); Female rats (161.0 to 194.0g)
- Fasting period before study: No
- Housing: in groups of up to five by sex in solid floor polysulphone cages with bedding bags.
- Diet: ad libitum 4 RF 21
- Water: ad libitum
- Acclimation period: three weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): approximately fifteen to twenty air changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light and twelve hours darkness cycle

IN-LIFE DATES: 23 March 2016 (allocation of animals) to 27 July 2016 (final day of necropsy)

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was prepared at appropriate concentrations in softened water. The stability of the test item formulations at the lowest and highest concentrations formulated (10 and 100 mg/mL) was determined and these formulations were found to be stable for up to 24 hours when stored at room temperature and up to 8 days when stored at 4°C. Formulations were prepared on a weekly or daily basis.
VEHICLE
- Concentration in vehicle: 0, 10, 30, 100 mg/mL (main phase) and 0, 100 mg/mL (recovery phase)
-Dose volume: 10 mL/kg bw (on the basis of the most recently recorded body weight)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the formulations prepared on Weeks 1 and 13 were analysed to confirm the test item concentration for all dosing solutions prepared. The results indicate that the prepared formulations were within 91.55% to 105.89% of the nominal concentration.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

only daily during dosing phase

Dose / conc.:

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

Remarks:

Vehicle control

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10 males and 10 females per dose. 5 males and 5 females were used for the recovery phase.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen based on information from a
preliminary study.
- Rationale for animal assignment: The animals were randomly allocated to treatment groups using a stratified randomisation procedure to give approximately equal initial group mea body weights.
- Recovery group: Control and high dose group included 5 additional animals per sex to be sacrificied after 4 weeks recovery

Positive control:

Not requried for this study type.

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS
All clinical signs were recorded for individual animals. Once before commencement of
treatment and at least once daily during the study, each animal was observed and any clinical
sign was recorded.

Observations were performed at the same time interval each day, the interval was selected
taking into consideration the presence of post-dose reactions.

Once before commencement of treatment and at least once per week from the start of treatment,
each animal was given a detailed clinical examination. Each animal was observed
in an open arena. The test included observation of changes in gait and posture, reactivity
to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and
effects on the autonomic nervous system (e.g. lachrymation, piloerection, unusual respiratory
pattern). Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and
excretions were also recorded.

BODY WEIGHT
Individual body weights were recorded on the day of allocation to treatment groups, the day before treatment commenced and at weekly intervals thereafter. Body weights were also recorded just prior to necropsy.

FOOD CONSUMPTION
The weight of food consumed by each cage of rats was recorded at weekly intervals following
allocation. The interval between allocation and treatment initiation was less than a full week.
The group mean daily intake per rat was calculated.

OPHTHALMOSCOPIC EXAMINATION
Both eyes of all animals were examined prior to the commencement of treatment by means of
an ophthalmoscope, and by a slit-lamp microscope, after the instillation of 0.5% Tropicamide
(Visumidriatic®, Visufarma, Rome, Italy). The eyes of all animals from high dose and control
groups were re-examined during Week 13 of treatment.
CLINICAL PATHOLOGY
Prior to necropsy, samples of blood were withdrawn under isofluorane anaesthesia from the
abdominal vena cava of 10 male and 10 female animals from each group, following overnight
food deprivation (approximately 17-18 hours).
Blood samples were collected and analysed in the same order. The blood samples collected
were divided into tubes as follows:
– EDTA anticoagulant for haematological investigations
– Heparin anticoagulant for biochemical tests
– Citrate anticoagulant for coagulation tests

HAEMATOLOGY
Parameters checked included: Red blood cell count, Haematocrit, Haemoglobin, Mean corpuscular haemoglobin, Mean red blood cell volume, Mean corpuscular haemoglobin concentration, White blood cell count, Differential leukocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils, large unstained cells), Platelets, Reticulocyte count

CLINICAL CHEMISTRY
Parameters checked included: Urea, Glucose, Total protein, Albumin, Globulin, Albumin/Globulin ratio, Sodium, Potassium, Chloride, Calcium, Phosphorus, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Gamma-glutamyltransferase, Creatinine, Total cholesterol, Total bilirubin, Triglycerides

COAGULATION
Prothrombin time was analysed.

NEUROTOXICITY
Once during Week 12 of treatment and once during Week 4 of recovery, an evaluation of
sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive
stimuli) and an assessment of grip strength were also performed.

MOTOR ACTIVITY
The motor activity (MA) of all animals was measured once during Week 13 of treatment and
once during Week 4 of recovery by an automated activity recording. Measurements were
performed using a computer generated random order.

Sacrifice and pathology:

GROSS PATHOLOGY
Animal in extremis and those that had completed the scheduled test period were killed
by exsanguination under isofluorane anaesthesia. All animals were subjected to necropsy,
supervised by a pathologist.

ORGAN WEIGHTS
The following organs, removed from animals that were killed at the test period, were dissected free from fat and weighed before fixation:
Adrenals, Brain (cerebrum, cerebellum, medulla/pons), Epididymides, Heart, Kidneys, Liver, Ovaries (including oviducts), Spleen, Testes, Thymus, Cervix,
The ratios of organ weight to body weight were calculated for each animal.

HISTOPATHOLOGY
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, Davidson’s fluid or modified Davidson’s fluid as appropriate:
Abnormalities, Adrenals, Aorta, Bone marrow (from sternum), Bone & bone marrow (sternum), Brain (cerebrum, cerebellum and medulla/pons), Caecum, Colon, Duodenum, Epididymides, Eyes, Femur with joint, Heart, Ileum, Jejunum including Peyer’s patches, Kidneys, Liver, Lungs (including mainstem bronchi), Lymph nodes (cervical and mesenteric), Mammary area, Oesophagus, Ovaries, Oviducts. Pancreas, Pituitary gland, Prostate gland, Rectum, Salivary glands, Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal column, Spinal cord, Spleen, Stomach, Testes , Thymus, Thyroid/Parathyroid, Trachea, Urinary bladder, Uterus (with cervix).

All tissues (with the exception of the eyes, femur, seminal vesicles, skeletal muscle, spinal column and oviducts which were not examined) were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Examination was conducted on tissues specified from all animals in the control and high dose groups dying during the treatment period or killed at the end of the 13 weeks of treatment, tissues from one animal killed during the treatment period and all abnormalities in all main phase groups.

Other examinations:

None

Statistics:

Standard deviations were calculated as considered appropriate. For continuous variables the significance of the differences amongst groups was assessed by analysis of variance.
Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If the data were found to be inhomogeneous, a Modified t test (Cochran and Cox) was applied.

The mean values, standard deviations and statistical analysis were calculated from the actual
values in the computer without rounding off. Statistical analysis of histopathological finding
was carried out by means of a nonparametric Kolmogorov-Smirnov test.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No signs of toxicological significance were recorded during treatment. Staining or damaged eye, hairloss and tooth broken/missing were recorded on occasion regardless treatment groups.
No signs were observed in animals during the recovery period. Weekly repeated observation of animals at removal from the cage and in an open arena did not show differences between groups. No differences were found during the recovery period.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Comparable values of body weight were recorded between groups across the overall dosing period. There was a slight decrement observed at the end of treatment which was due to the overnight fasting for clinical pathology. No differences in body weight were recorded between the high dose and control animals during the recovery phase. No differences were observed in terminal body weight between groups both in males and females at the end of the dosing phase. No significant differences were observed in terminal body weight between control and high dose groups at the end of the recovery phase.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant lower food consumption was recorded in the mid- and high dose females on Day 92. No toxicological relevance was given to this change. The food consumption was reduced in all groups including controls during the last week of treatment. No differences were seen in males. Food consumption did not differ between the control and high dose groups during the recovery phase.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No findings were detected in both eyes of all animals examined before the start of treatment. No abnormalities were found in the eyes of the control and high dose animals re-examined
at the end of the dosing phase (Week 13 of treatment). No ophthalmoscopy was carried out in recovery animals.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Minimal increase of erythrocytes, haemoglobin and haematocrit was recorded in males dosed with 1000 mg/kg bw/day and in females receiving 300 mg/kg bw/day. In addition, mean corpuscular haemoglobin concentration was significantly lower than controls, at statistical analysis, in females dosed at 300 mg/kg bw/day. Due to the minimal severity (3% to 7%) and/or absence of dose-relationship, the above changes were considered irrelevant. No changes were recorded in the coagulation test.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant fluctuations of some biochemical parameters were recorded in treated animals, mainly males. Due to the low severity, the inconsistency between sexes and/or the absence of dose-relation, these changes were considered of no toxicological significance. One control male showed very high values liver enzymes such as alanine aminotransferase
(ALT) and aspartate aminotransferase (AST). However, no other related-findings of possible liver injury were recorded. No anomalies were recorded during the analysis, therefore these data were considered acceptable even if no pathological explanation was found.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

The hindlimb landing foot splay measurement was significantly lower in males of the high dose group compared to controls. No differences were found in females. Although an increase in landing foot splay (the distance of the two resulting ink spots) has been reported for compounds that produce motor dysfunction, the reduced values did not clearly define a neurotoxicity effect of the test compound. Additionally, grip strength was found significantly lower in males of the high dose group compared to controls. Although this test is quite sensitive in detecting neuromuscular impairments, all detailed functional battery of observations (FOB) such as changes in gait, posture, rearing (e.g decreased rearing), bizarre behaviour or changes in motor response to a variety of sensory stimuli, did not show differences between groups. Furthermore, the quantitative measures of motor activity did not reveal differences between groups, indicating that spontaneous locomotor activity of the animals was not affected by treatment. Therefore, the significantly reduced landing foot splay and grip strength in males of the high dose group were considered of doubtful significance to identify neurofunctional deficits. Furthermore, these data were inconsistent between sexes. A significantly higher grip strength value was recorded in males of the high dose group in the recovery phase. The mean value was approximately 2.5-fold higher than the control group with no clear toxicological significance. No other relevant changes were detected. The statistically reduced motor activity in high dose females was considered incidental.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Significantly increased absolute and relative adrenal and relative kidneys weights were recorded in high dose males at the end of the dosing phase. Relative kidneys and liver weights were significantly increased in high dose females. In the absence of relevant changes at clinical pathology or at histological examination, these findings were considered of no toxicological relevance. No other changes were recorded. Significantly higher absolute spleen weight was recorded in high dose males compared to controls at the end of the recovery phase. This difference was minimal and not considered of toxicological relevance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No remarkable changes were noted at post mortem examination in treated animals at the end of treatment and after 4 weeks of recovery period, when compared with controls.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were noted. All observed lesions are known to occur spontaneously in Sprague Dawley SD rats of the same age and/or have a comparable incidence in the control group.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Key result

Critical effects observed:

no

Summary of significant findings

	Males				Females
Dose (mg/kg bw/d)	0	100	300	1000	0	100	300	1000
Mean food consumption (Day 92) (g)	21.37	19.80	20.51	21.42	17.91	17.09	15.39^+D	14.82^+D
Mean landing foot splay (Day 78-males; Day 79-females) (cm)	5.653	5.590	5.525	4.577^*D	4.113	4.440	4.180	3.287
Mean grip strength (Day 78-males; Day 79-females) (g)	9.163	7.470	10.435	6.448^*D	9.727	10.585	9.425	9.883
Mean grip strength (Recovery day 25) (g)	5.310	N/A	N/A	11.980^+D	10.370	N/A	N/A	9.340
Weight of adrenal glands (g)	0.0477	0.0484	0.0501	0.0576^+D	0.0576	0.0614	0.0607	0.0597
Adrenal glands weight to terminal body weight (% of terminal body weight)	0.0107	0.0111	0.0116	0.0138^+D	0.0213	0.0224	0.0221	0.0222
Kidney weight to terminal body weight (% of terminal body weight)	0.6419	0.6543	0.6301	0.7171^+D	0.6286	0.6404	0.6326	0.6746^*D
Liver weight to terminal body weight (% of terminal body weight)	2.5677	2.4388	2.4487	2.6044	2.2860	2.2610	2.3624	2.5048^*D
Weight of spleen (Recovery group) (g)	0.8348	N/A	N/A	0.9344^*D	0.7030	N/A	N/A	0.6656

+D Statistically significant at the 0.01 level with Dunnett LSD Test
*D Statistically significant at the 0.05 level with Dunnett LSD Test
N/A Not applicable

Conclusions:

A NOAEL of 1000 mg/kg bw/d can be determined for this study, in the absence of any adverse effects at the highest dose level.

Executive summary:

In a 90-day study performed to OECD 408, TMPO was administered by gavage to groups of Sprague Dawley rats (10/sex) on ninety-two consecutive days at dose levels of 100, 300 and 1000 mg/kg bw/d. A control group was administered with the vehicle (softened water) by oral gavage. Additional animals (5/sex) were treated with TMPO for ninety-two consecutive days at a dose level of 1000 mg/kg bw/d followed by a 4-week recovery period. A control recovery group was administered with the vehicle by oral gavage. Clinical signs, functional observations, body weight change, dietary intake, ophthalmoscopy, were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed. There were no unscheduled deaths during the study. There were no treatment-related clinical signs observed during the dosing or recovery phases. No treatment-related changes in body weight or terminal body weight were observed, although there was a small statistically significant decrease in food consumption on Day 92 at 300 and 1000 mg/kg bw/d in females only. An increase in absolute and relative weight of the adrenal glands was observed in the high dose group in males only. An increase in the relative weight of the liver was observed in the high dose group in females only. In addition, an increase in the relative weight of the kidneys was observed in the high dose group of both sexes. Since no changes were noted following histopathological examination, these were considered to be of no toxicological relevance. At the end of the recovery phase, an increase in the relative weight of the kidney was observed in the high dose group in males only. Since these increases were only slight, these were also considered to be of no toxicological relevance. Following the Functional Observation Battery, a statistically significant decrease in the landing foot splay and a statistically significant increase in mean grip strength was observed in the high dose group in males only, with the latter also being seen in the recovery group. In the absence of any other changes and the inconsistencies between sexes, this was considered not to be a clear indication of any neurofunctional deficits. Some changes in clinical chemistry were noted. Changes in urea, total bilirubin, creatinine, chloride, sodium, phosphorus, albumin and albumin/globulin ratio in males only, alanine aminotransferase in females only and glucose in both sexes were recorded. Since these were inconsistent between sexes and/or showed no clear indication of a concentration related response, these were considered to be of no toxicological significance. Haematological changes were observed at 1000 mg/kg bw/d in males only and at 300 mg/kg bw/d in females only. These changes were considered to be irrelevant since the changes were slight (3-7% change) and showed no clear evidence of a concentration related response. There were no ophthalmological, pathological or histopathological changes which were treatment related. A NOAEL of 1000 mg/kg bw/d can be determined for this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

High quality studies of repeated dose toxicity are available for the submission substance.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The sub-acute oral repeated dose toxicity of 3-ethyloxetane-3-methanol has been investigated in a 28-day study in rats conducted according to OECD Test Guideline 407 (Sawaki, 1997). In the study groups of male and female Crj: CD (SD) rats (6/sex/group) were administered the test substance oxetane (OH) by oral gavage at 0, 40, 200 or 1000 mg/kg bw daily for 28 days, followed by a 14-day recovery period. No mortalities were observed in any of the treated or control animals. No treatment-related effects were observed in clinical signs, body weight and food intakes during the dosing period and in the haematological and blood chemical examinations, urinalysis, organ weights, necropsy and histopathological examinations at the termination of the dosing period. No treatment-related effects were observed in animals during the post-exposure recovery period. It was concluded that the NOAEL of 3-ethyloxetane-3-methanol for sub-acute repeated dose toxicity in rats was 1000 mg/kg bw/day (e.g. the highest dose tested).

In a 90-day study performed to OECD 408, TMPO was administered by gavage to groups of Sprague Dawley rats (10/sex) on ninety-two consecutive days at dose levels of 100, 300 and 1000 mg/kg bw/d. A control group was administered with the vehicle (softened water) by oral gavage. Additional animals (5/sex) were treated with TMPO for ninety-two consecutive days at a dose level of 1000 mg/kg bw/d followed by a 4-week recovery period. A control recovery group was administered with the vehicle by oral gavage. Clinical signs, functional observations, body weight change, dietary intake, ophthalmoscopy, were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed. There were no unscheduled deaths during the study. There were no treatment-related clinical signs observed during the dosing or recovery phases. No treatment-related changes in body weight or terminal body weight were observed, although there was a small statistically significant decrease in food consumption on Day 92 at 300 and 1000 mg/kg bw/d in females only. An increase in absolute and relative weight of the adrenal glands was observed in the high dose group in males only. An increase in the relative weight of the liver was observed in the high dose group in females only. In addition, an increase in the relative weight of the kidneys was observed in the high dose group of both sexes. Since no changes were noted following histopathological examination, these were considered to be of no toxicological relevance. At the end of the recovery phase, an increase in the relative weight of the kidney was observed in the high dose group in males only. Since these increases were only slight, these were also considered to be of no toxicological relevance. Following the Functional Observation Battery, a statistically significant decrease in the landing foot splay and a statistically significant increase in mean grip strength was observed in the high dose group in males only, with the latter also being seen in the recovery group. In the absence of any other changes and the inconsistencies between sexes, this was considered not to be a clear indication of any neurofunctional deficits. Some changes in clinical chemistry were noted. Changes in urea, total bilirubin, creatinine, chloride, sodium, phosphorus, albumin and albumin/globulin ratio in males only, alanine aminotransferase in females only and glucose in both sexes were recorded. Since these were inconsistent between sexes and/or showed no clear indication of a concentration related response, these were considered to be of no toxicological significance. Haematological changes were observed at 1000 mg/kg bw/d in males only and at 300 mg/kg bw/d in females only. These changes were considered to be irrelevant since the changes were slight (3-7% change) and showed no clear evidence of a concentration related response. There were no ophthalmological, pathological or histopathological changes which were treatment related. A NOAEL of 1000 mg/kg bw/d can be determined for this study.

Justification for classification or non-classification

The available data indicate that 3-ethyloxetane-3-methanol does not cause systemic or target organ toxicity after repeated oral dosing in rats. The substance does not meet the criteria for classification for repeated dose toxicity according to the CLP Regulation.