4-(5-(3,5-dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydro-1,2-oxazol-3-yl)-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Start: 24 June 2016 (animal arrival), End: 17 October 2016 (foetal pathology)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This developmental toxicity study was generated to meet the data requirements of regulations not related to REACH in non-EEA countries.

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)

Version / remarks:

2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-(5-(3,5-dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydro-1,2-oxazol-3-yl)-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide

Cas Number:

2061933-85-3

Molecular formula:

C23H19Cl2F4N3O4

IUPAC Name:

4-(5-(3,5-dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydro-1,2-oxazol-3-yl)-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide

Test material form:

solid: particulate/powder

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Source: Envigo, Hillcrest, Dodgeford Lane, Belton, Loughborough, Leicestershire, LE11 4TE, England
Age: approximately 4 months
Weight: 2.79 to 4.21 kg
Acclimatisation: at least 2 days
Housing: Individually in perforated-floor cages suspended over paper-lined trays.
Diet: STANRAB (P) SQC ad libitum
Water: Mains tap water ad libitum
Temperature: 16 to 23 °C
Humidity: 49 to 91%
Photoperiod: alternating 12-hour light and darkness cycles

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% (w/v) aqueous carboxymethylcellulose and 0.1% (v/v) Tween 80

Details on exposure:

All doses were administered once daily (orally), on gestation days 6 to 27, in a volume of 2 mL/kg bodyweight/day. Dosing was based on individual body weight at the time of dosing.

The test item was formulated every 7 to 8 days, within the known stability period, for each group separately, as a suspension in 0.5% (w/v) aqueous carboxymethylcellulose and 0.1% (v/v) Tween 80. A weighed quantity of test item was added to a mortar, wetted with a small quantity of vehicle and initially made into a smooth paste using a pestle. After further addition of vehicle and mixing, the resultant suspension was transferred into a tared beaker on a balance. The mortar was thoroughly rinsed out with vehicle and added to the suspension which was then made up to final weight with vehicle and mixed with a laboratory homogeniser. The formulation was then divided into daily aliquots for dosing and stored refrigerated (2 °C to 8 °C).
Formulations were removed from refrigerated storage and stirred for at least 15 minutes before the start of dosing and until completion of dosing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to the start of the study, stability of the test substance in 0.5% (w/v) aqueous carboxymethylcellulose and 0.1% (v/v) Tween 80 had been assessed and formulations over a concentration range of 0.1, 1 and 20 mg/mL, were shown to be homogeneous and stable for up to 7 days at room temperature, up to 13 days when stored refrigerated and for 1 month when stored frozen (approximately -18 ºC).

Samples were taken from each test item formulation prepared. Samples prepared for the first day of dosing were analysed for the substance using a validated method to confirm homogeneity and achieved concentrations. Having satisfactorily confirmed homogeneity for the first day of dosing, samples were taken from all test item formulations prepared for use towards the end of the dosing period and analysed to confirm concentration only. Samples were taken on these days from the vehicle used to dose Controls and were analysed to confirm absence of test item.

Details on mating procedure:

Each female had been mated with a sexually mature male of the same strain and given an intravenous injection of 25IU Luteinising Hormone (LH) to stimulate ovulation. The day of mating was designated Day 0 of gestation.

Duration of treatment / exposure:

Gestation days 6 to 27

Frequency of treatment:

Daily administration

Duration of test:

Dams were sacrificed on gestation day 28

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

3.5 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

7.5 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

15 mg/kg bw/day (nominal)

No. of animals per sex per dose:

22 sexually mature tim-mated females

Control animals:

yes, concurrent vehicle

Details on study design:

Animals were randomly assigned to groups using a stratified body weight recorded on Day 0 of gestation, ensuring females paired with the same male were distributed across the groups randomly.
The dose levels were selected on the basis of results from a dose range finding study performed at test facility. The reduction in body weight gain observed at the high dose level of 15 mg/kg/day in the preliminary study represented a suitable degree of maternal toxicity for this dose level to be selected as the high dose in this study. Dose levels of 7.5 and 3.5 mg/kg/day were selected as the intermediate and low doses respectively, in order to demonstrate dose response.

Examinations

Maternal examinations:

Mortality and morbidity was observed twice daily with individual records.
Clinical signs were observed daily with individual records.
Body weight: recorded daily from day 4 to 28 of gestation
Food consumption: amount of food consumed by each animal was recorded daily from day 4 to 6 of gestation, then every second day.
Dams sacrificed on day 28 of gestation were subjected to post-mortem examinations, including record of dead body weight, investigation of thoracic and abdominal cavities, major organs and uterus. Organs or tissues showing any macroscopic abnormalities were recorded and retained.

Ovaries and uterine content:

Gravid uterus and placenta weights were recorded. The number of corpora lutea and the number and distribution of implantations for each female was recorded and the uterus of any apparently non-pregnant female were stained to confirm pregnancy status.

Blood sampling:

Not applicable

Fetal examinations:

Approximately 50% of the foetuses in each litter were decapitated and the heads fixed in Bouin's fluid and examined by serial sectioning. The intact foetuses and bodies of the decapitated foetuses were briefly placed in alcohol and subjected to micro-dissection, where the viscera were examined, the sex recorded and the foetuses eviscerated. A coronal section was made through the head of the intact foetuses along the frontal parietal suture and the brain examined. All carcasses were subsequently cleared in potassium hydroxide and stained with Alizarin red S and Alcian blue to visualise the ossified skeleton and cartilage and examined.

Statistics:

General Approach: All statistical tests were two-sided with minimum significance levels of 5% and 1%. Non-parametric statistics were not routinely conducted. The litter, rather than the foetus, was considered as the experimental unit. When used, Dunnett’s test was conducted regardless of the outcome of the analysis of variance (ANOVA) or analysis of covariance (ANCOVA).

Data were examined for unusually high or low values which could influence the statistical analysis and interpretation (possible outliers). After examining for any outliers, if the variances were clearly heterogeneous, transformations (e.g. log, double arcsine or square root) were used in an attempt to stabilise the variances. If the transformations failed, the data set was examined and a decision taken on further action.

For Quantitative Data: Body weight, cumulative body weight gain from the start of dosing, food intake, terminal body weight, numbers of corpora lutea, implants, live foetuses, dead foetuses, early deaths, late deaths, gravid uterus weight, total litter weight, placental weight and mean foetal weight (sexes separately and combined) were analysed using a parametric ANOVA.

For Percentages: Pre-implantation loss, post-implantation loss, sex ratios (% male foetuses) and litter based mean percentages were analysed using a parametric ANOVA, following a double arcsine transformation (Freeman and Tukey, 1950).

Maternal Performance: (e.g. the proportion of females with live foetuses at termination, abortions, total resorptions) were analysed by a two-tailed Fisher’s Exact Text (Steel and Torrie, 1980), comparing each treated group to the control group.

Foetal Morphology Data: The incidence of foetal malformations and developmental variations (external, visceral and skeletal) were summarised as the proportion of foetuses affected, the proportion of litters affected and the proportion of foetuses affected within each litter. The proportions of litters affected were analysed by

Indices:

Pre-implantation loss (%) = (number of corpora lutea - number of implantation sites)/number of corpora lutea x 100
Post-implantation loss (%) = (number of implantation sites - number of live foetuses)/number of implantation sites x 100
Mean pre- and post-implantation losses were calculated on a proportional litter basis.
Mean foetal body weights were calculated separately by sex for each litter and group means were calculated from the litter means.
The percentage of foetuses in each litter exhibiting each classification of abnormality was calculated; group mean percentages were calculated from the litter percentages.
The percentage of male foetuses, out of the total number of foetuses, was calculated for each litter.

Historical control data:

Historical control data on pregnancy and litter data obtained in the laboratory over the period from 2012 and 2015 were provided in the report.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Description (incidence):

Two incidental deaths occurred in the study, which were considered to be due to an accidental dosing trauma.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no statistically significant differences from Control in body weight or body weight gain in any dose group. At 15 mg/kg/day, body weight gains were generally lower than the Control group, with overall gain for the dosing period 9% lower than the Controls. This is consistent with the results of a preliminary study in pregnant rabbits, where a dose level of 15 mg/kg/day resulted in a 14% lower weight gain compared to controls.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not specified

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Head: open eyes, uni- or bilateral
Brain: exencephaly
Spine: cervical, thoracic, lumbar or sacral cord: spina bifida
Umbilicus: omphalocele
Tail: filamentous
Forelimb: severe abnormal flexure, uni- or bilateral malrotation
Hindlimb: uni- or bilateral malrotation
The nature and intergroup distribution of these major foetal abnormalities do not indicate an adverse effect of the substance. They were isolated, with a low incidence of each type of abnormality and were either disparate, lacked a dosage-related distribution, were within the current historical control data range or are known to be seen spontaneously in rabbits of this strain in these laboratories.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skull: interparietal absent, occipital absent
Cervical vertebrae: one or more centra absent, one or more neural arches absent, odontoid process absent
Thoracic vertebrae: one or more centra fused severely, one or more neural arches fused severely, right additional neural arch ossified, one or more cartilaginous centra fused severely
Lumbar vertebrae: one or more centra severely fused, one or more hemicentra absent
Sacral vertebrae: one or more centra severely fused, one or more neural arches absent
Caudal vertebrae: one or more centra severely fused, one or more neural arches severely fused or absent
Sternum: one or more sternebrae severely fused
Pelvic girdle: severe asymmetric insertion
The nature and intergroup distribution of these major foetal abnormalities do not indicate an adverse effect of the substance. They were isolated, with a low incidence of each type of abnormality and were either disparate, lacked a dosage-related distribution, were within the current historical control data range or are known to be seen spontaneously in rabbits of this strain in these laboratories.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Uterine horn: uni- or bilateral absence
Ovary: uni- or bilateral absence
Liver: diaphragmatic hernia in one or more lobes
The nature and intergroup distribution of these major foetal abnormalities do not indicate an adverse effect of the substance. They were isolated, with a low incidence of each type of abnormality and were either disparate, lacked a dosage-related distribution, were within the current historical control data range or are known to be seen spontaneously in rabbits of this strain in these laboratories.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

changes in postnatal survival

external malformations

skeletal malformations

visceral malformations

Fetal abnormalities

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

external: eye

external: limb

external: tail

skeletal: forelimb

skeletal: sternum

skeletal: rib

skeletal: vertebra

skeletal: pelvic girdle

skeletal: hindlimb

visceral/soft tissue: hepatobiliary

visceral/soft tissue: female reproductive system

Description (incidence and severity):

The nature and intergroup distribution of these major foetal abnormalities do not indicate an adverse effect of the substance. They were isolated, with a low incidence of each type of abnormality and were either disparate, lacked a dosage-related distribution, were within the current historical control data range or are known to be seen spontaneously in rabbits of this strain in these laboratories.

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

The No Observed Adverse Effect Level (NOAEL) for maternal toxicity and embryo-foetal development was considered to be 15 mg/kg/day.

Executive summary:

The prenatal developmental toxicity of the substance in rabbits was studied under GLP to OECD TG 414. Four groups of 22 sexually mature timed-mated female New Zealand White rabbits were administered 0 (vehicle control, 0.5% (w/v) carboxymethylcellulose with 0.1% (v/v) Tween 80), 3.5, 7.5 or 15 mg/kg/day by oral gavage, once daily, at a dose volume of 2 mL/kg body weight from Day 6 to Day 27 of gestation.
There were no deaths or clinical observations considered related to the test substance. At 15 mg/kg/day, body weight gain over the dosing period was 9% lower than in the control group. In animals given 3.5 or 7.5 mg/kg/day, body weight gains were similar to the controls and at all dose levels, there was no effect of the substance on overall mean food intake.
Pregnancy data were similar in all groups, with no adverse effect of the substance on the mean numbers of implantations, the incidences of pre- and post-implantation loss or on the mean number of live foetuses. Mean foetal and placental weights and foetal sex ratio were unaffected by the substance.
There was no adverse effect of the substance on the incidences of major, minor or variant foetal abnormalities.
Administration of the substance to pregnant New Zealand White rabbits at 3.5, 7.5 or 15 mg/kg/day, once daily by oral gavage from Day 6 to Day 27 of gestation, was well tolerated, with only slight decreases in body weight gain at 15 mg/kg/day indicating minor maternal toxicity at the highest dose tested. There was no adverse effect on pregnancy or embryonic or foetal development. On this basis, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity and embryo-foetal development was considered to be 15 mg/kg/day.