3-ethyloxetane-3-methanol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 March 2016 to 03 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

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:

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
Appearance Colourless liquid
RTC number 14802

Test animals

Species:

rat

Strain:

Sprague-Dawley

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: approx. 8 weeks for females; approx. 15 weeks for males
- Weight of female rats at Day 0 of gestation: 192.2 to 251.7
- Fasting period before study: No
- Housing: in groups of up to five by sex in solid floor polysulphone cages with bedding bags and nesting material (as necessary) before and after mating. During the mating period, rats were housed on the basis of 1 male to 1 female in polysulphone cages with a stainless steel mesh lid and floor.
- Diet: ad libitum 4 RF 21
- Water: ad libitum
- Acclimation period: two 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: 22 March 2016 (first day of mating) to 22 April 2016 (final day of necropsy).

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test item was administered orally by gavage at a dose volume of 10 mL/kg body weight. Control animals received the vehicle alone at the same dose volume.
The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The proposed formulation procedure for the test item was checked in the range from 10 to 100 mg/mL by chemical analysis (concentration) in RTC Study No. A2036 to confirm that the method was suitable. Final results for all levels were within 90-110% of nominal.
Samples of the formulations prepared on Weeks 1 and 4 were analysed to check the concentration.
Results of the analyses were within 90-110% of nominal.

Details on mating procedure:

The females were paired with male rats. Females were paired one to one in the home cage of
the male and left overnight. Vaginal smears were taken daily in the morning from the day
after pairing until a positive identification of mating was made. The day of mating, as judged
by the presence of sperm in the vaginal smear or by the presence of a copulation plug, was
considered as Day 0 of gestation (or Day 0 post coitum).

Duration of treatment / exposure:

All animals were dosed from Day 6 through Day 19 post coitum.

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.:

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

Doses / concentrations 3

Dose / conc.:

300 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:

Each group comprised 24 mated females.

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment: The animals were randomly allocated to treatment groups using a stratified randomisation procedure to give approximately equal initial group mean body weights.
- Recovery group: Control and high dose group included 5 additional animals per sex to be sacrificed after 4 weeks recovery

Examinations

Maternal examinations:

Mortality: Throughout the study, all animals were checked early in each working day and again in the
afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

Clinical signs: All clinical signs were recorded for individual animals. Each animal was observed daily and any clinical signs recorded starting from allocation until sacrifice.

Bodyweights: All animals were weighed on Days 0, 6, 9, 12, 15 and 20 post coitum.

Food consumption: Food consumption was measured on Days 6, 9, 12, 15 and 20 post coitum starting from Day 0 post coitum.

The animals were euthanised with carbon dioxide on Day 20 post coitum and necropsied.

The clinical history of the animals was studied and a detailed post mortem examination was
conducted (including examination of the external surface and orifices). Changes were noted
and the abnormalities preserved in 10% neutral buffered formalin.

Ovaries and uterine content:

The ovaries and uteri were examined to determine:
– Gravid uterine weight;
– number of corpora lutea;
– number of implantation sites;
– number, sex and weight of all live foetuses;
– number and sex of dead foetuses (foetuses at term without spontaneous movements
and breathing);
– number of intra-uterine deaths;
– gross evaluation of placentae.
Intra-uterine deaths were classified as:
– Early resorptions: only placental remnants visible.
– Late resorptions: placental and foetal remnants visible.
Uteri or individual uterine horns without visible implantations were immersed in a 20%
solution of ammonium sulphide to reveal evidence of embryonic death at very early stages
of implantation.

Fetal examinations:

Examination of foetuses: All live foetuses were examined externally.

Euthansia: All foetuses were sacrificed by intraperitoneal injection of Sodium Thiopental followed by hypothermia.

Approximately one-half of the foetuses (i.e., routinely, every second live foetus) in each litter was preserved in Bouin’s solution for subsequent fixed-visceral examination. The remaining foetuses were eviscerated after which the carcasses were fixed in 95% (v/v) ethanol for subsequent skeletal examination. Skeletal and fixed-visceral examinations were performed in all groups.
Structural deviations were classified as follows:
Malformations - Major abnormalities that are rare and/or affect the survival or health of the species under investigation.
Anomalies - Minor abnormalities that are detected relatively frequently.
Variants -A change that occurs within the normal population under investigation and is unlikely to
adversely affect survival or health. This might include a delay in growth or morphogenesis that would have otherwise followed a normal pattern of development.

Sex ratios of the foetuses were calculated as the percentage of males per litter. All derived values (e.g., means, percentages, ratios) were first calculated within the litter and
the group values derived as a mean of individual litter values. Foetal structural deviations were expressed as the percentage of affected foetuses relative to all foetuses examined per group, as well as in terms of the mean litter percentage of affected litters.

Statistics:

For continuous variables the significance of the differences amongst group means was
assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of non-continuous variables was carried out by means of the Kruskal-
Wallis test.

Indices:

Pre-implantation loss was calculated as a percentage from the formula:
Pre-implantation loss = (number of corpora lutea minus number of implantations)/ number of corpora lutea
Expressed as a percentage.

Post-implantation loss was calculated as a percentage from the formula:
Post-implantation loss = (number of implantations minus number of live foetuses)/ number of implantations
Expressed as a percentage.

Total implantation loss was calculated as a percentage from the formula:
Total implantation loss = (number of corpora lutea minus number of live foetuses)/number of corpora lutea
Expressed as a percentage.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No signs of toxicological significance were noted during the study and no signs of reaction to
treatment were noted during the dosing period.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No animal died during the study. One female in the control group gave premature birth on
gestation Day 20. Two females in the low dose group were found not pregnant at necropsy.
The number of females with live foetuses on gestation Day 20 was 23 in the control group, 22
in the low dose group and 24 in mid- and high dose groups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No differences in body weight were noted between control and treated groups. At body
weight gain, a statistically significant decrease of approximately 17% was noted on Day 12
post coitum in mid-dose females when compared to controls. The change was considered to
be without toxicological relevance.

No differences in terminal body weight were observed in treated groups compared to the control group.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No differences in food consumption were noted between control and treated groups. The
statistically significant decrease of approximately 7% detected on Day 9 post coitum in high
dose females was considered to be without toxicological relevance.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No differences in gravid uterus weight and absolute weight gain were
observed in treated groups compared to the control group.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were observed at post mortem examination in treated animals,
when compared to the controls.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

not specified

Early or late resorptions:

not specified

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

no effects observed

Other effects:

not specified

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratios were not affected by treatment.

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

No significant effects were seen in the litter data even if a slight trend towards a decrease in mean foetal weight and in litter weight (up to 6%) was seen in the mid- and high dose groups.

Changes in postnatal survival:

not examined

External malformations:

effects observed, treatment-related

Description (incidence and severity):

A total of 9 small foetuses (< 2.7 g) were detected; 4 out of 325 in the mid-dose group and 5 out of 320 in the high dose group. In addition, abnormal dark red area was detected on muzzle and sacral region of one small foetus in the high dose group. These findings were considered incidental.
No others abnormalities were detected at the external examination of foetuses.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Skeletal observations of the low dose foetuses were comparable to controls. An increased incidence of no ossification of metacarpals (4th), incomplete or no ossification of sternal elements (5th and 6th), as well as incomplete or no ossification of the centrum of the thoracic vertebrae, were seen in mid- and high dose groups compared to controls. In addition, incomplete or no ossification of the pubis, associated with incomplete ossification of the ischium, was seen in few small foetuses in the same treated groups. The findings noted are considered to be due to a slight foetal underdevelopment also demonstrated by the presence of small foetuses in mid- and high dose groups.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Visceral observations of the low dose foetuses were comparable to controls. Single cases of malformations were detected in control, mid- and high dose groups. Extremely enlarged uterers were seen in 1 foetus each in the control, mid- and high dose groups; extreme pelvic dilatation of kidney was seen in 2 foetuses from different litters in the mid-dose group and in 1 foetus in the high dose group; 1 foetus in the high dose group showed hepatomphalocele, and another one showed hepatic lobe agenesis. No clear treatment relationship was attributed to these findings due to their low incidence. Anomalies were detected in a few foetuses in all groups, with lower or similar incidence in treated groups respect to controls. Therefore they were considered to be without toxicological significance.

Other effects:

no effects observed

Effect levels (fetuses)

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 absence of adverse toxic effects

Fetal abnormalities

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: sternum

skeletal: vertebra

skeletal: pelvic girdle

Description (incidence and severity):

Minor effects on skeletal ossification, secondary to low foetal weight and not considered to be adverse.

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

External and skeltal examination of foetuses

Dose (mg/kg bw/d)	0 (control)	100	300	1000
Small foetuses	0/308	0/306	4/325	5/325
Mean litter weight (g)	50.54	50.54	48.82	47.49
Mean foetal weight (g)	3.78	3.65	3.59	3.56
No ossification of metacarpals (4^th)	29/162	30/158	61/170	51/169
Incomplete or no ossification of sternal elements (5^th)	18/162	34/158	56/170	49/169
Incomplete or no ossification of sternal elements (6^th)	20/162	30/158	52/170	54/169
Incomplete or no ossification of the centrum of the thoracic vertebrae	15/162	14/158	25/170	35/169
Incomplete or no ossification of the pubis	0/162	0/158	3/170	7/169

Fate of females, maternal weight and litter data

Dose (mg/kg bw/d)	0 (control)	100	300	1000
Mated (#)	24	24	24	24
Non-pregnant (#)	0	2	0	0
Premature birth (#)	1	0	0	0
Litters examined (#)	23	22	24	24
Terminal weight (g)	377.94	379.12	374.63	373.13
Weight gain (g)	72.44	70.53	70.44	70.50
Corpora lutea (#)	14.26	14.41	14.50	14.25
Implantations (#)	13.78	14.14	14.33	13.79
Early uterine death (#)	0.39	0.23	0.75	0.46
Late uterine death (#)	0.00	0.00	0.04	0.00
Viable young (#)	13.39	13.91	13.54	13.33

Applicant's summary and conclusion

Conclusions:

A NOAEL of 1000 mg/kg bw/d for maternal and developmental toxicity can be determined for this study.

Executive summary:

In a study conducted in accordance with OECD TG 414, TMPO was dosed via oral gavage to groups of 24 mated Sprague Dawley female rats at dose levels of 100, 300 and 1000 mg/kg bw/d on GD 6 -19. A control group comprising 24 mated females was administered with the vehicle (softened water) by oral gavage. Clinical signs, bodyweight and food consumption were monitored throughout the treatment phase. All maternal animals were caesarean sectioned on Day 20 post coitum and subjected to gross necropsy, including the ovaries and uteri. All live foetuses were examined externally. Approximately half of the foetuses were preserved for subsequent fixed-visceral examination and the remaining foetuses fixed for subsequent skeletal examination. There were no unscheduled deaths during the study. One female in the control group gave premature birth on gestation Day 20 and two females in the low dose group were not pregnant at necropsy. There were no clinical signs, changes in body weight, body weight gain, food consumption, terminal body weight, uterus weight and absolute weight gain which were significant in the treated groups when compared to the control group. There was a slight trend towards a decrease in mean foetal weight and in litter weight in the mid- and high-dose groups. Some small foetuses (less than 2.7 g) were detected in the mid- and high dose groups which was considered to be incidental. There were no treatment related findings following the visceral examination of the foetuses. Some skeletal changes which were defined as variants or anomalies were observed in higher incidences in mid- and high dose groups. These changes were attributed to the decreases in mean foetal weight and litter weight or associated to growth retardation in the small foetuses. The No Observed Adverse Effect Level (NOAEL) was determined to be 1000 mg/kg bw/d.