Bis[2-[2-(1-methylethyl)-3-oxazolidinyl]ethyl] hexan-1,2-diylbiscarbamate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-06-07 to 2016-06-30

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)

Version / remarks:

22 January 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Bis[2-[2-(1-methylethyl)-3-oxazolidinyl]ethyl] hexan-1,2-diylbiscarbamate

EC Number:

261-879-6

EC Name:

Bis[2-[2-(1-methylethyl)-3-oxazolidinyl]ethyl] hexan-1,2-diylbiscarbamate

Cas Number:

59719-67-4

Molecular formula:

C24H46N4O6

IUPAC Name:

bis[2-(2-isopropyl-1,3-oxazolidin-3-yl)ethyl] hexane-1,6-diylbiscarbamate

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt., 1103 Budapest ,Cserkesz u. 90., Hungary
- Age at study initiation: 11 - 12 weeks
- Weight at study initiation: 199 - 230 g
- Housing: 2 - 3 females per cage
- Diet: ad libitum, ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" (ssniff Spezialdiäten GmbH, D-59494 Soest, Germany)
- Water: ad libitum, tap water
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 23
- Humidity (%): 44 - 68
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

water-free PEG 400

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle (PEG 400) in concentrations of 200 mg/mL, 60 mg/mL and 20 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility and stored at 5 ± 3 °C until use but not longer than for one day.

VEHICLE
- Justification for use and choice of vehicle: The test item hydrolyses very quickly in water therefore PEG 400 was used for preparing formulations appropriate for oral administration. PEG 400 was a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 20, 60, 200 mg/mL
- Amount of vehicle: 5 mL/kg bw
- Lot/batch no.: 14B110500, 15I040501

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of dosing solutions (control of concentration and homogeneity) was performed in the Analytical Laboratory of Test Facility two times during the study. Five samples from different places were taken from each concentration for analysis of concentration and homogeneity on two occasions. Similarly, five samples were taken from the vehicle (Group 1) and analysed.
The suitability of the chosen vehicle for the test item was analytically proven. Recovery was between 102 and 104 % of nominal concentrations at 10 and 200 mg/mL in PEG 400, respectively. Incozol 4 proved to be stable at room temperature for 4 hours and at 5 ± 3 °C for one day. A separate analytical report provided these results (Toxi-Coop study no. 644-100-1325).

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 : 1-3
- Length of cohabitation: two to four hours
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

Day 5 to Day 19 of Gestation

Frequency of treatment:

daily

Duration of test:

14 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

23 to 27 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose setting was based on findings obtained in a previous repeated dose toxicity study with Incozol 4 in the Rat (A 28-Day Repeated Dose Oral Toxicity Study of CG-S4 in Rats; Study no. TBH-1460 (KG-2011-412); GLP and 90-Day Oral Gavage Toxicity Study with Incozol 4 in the Rat; Study no.: 644-408-1326) and in agreement with the Sponsor.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day
- Cage side observations included: check of behavior and general condition, duration and severity of the clinical signs

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: on gestation days 0, 3, 5, 8, 11, 14, 17 and 20

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule: between gestation days 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20 by re-weighing the non-consumed diet

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: uterus, ovaries, viscera in general

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No

Statistics:

Data were individually recorded on data sheets, transferred, and compiled by computer or compiled manually.
The statistical evaluation of data was performed with the program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity is detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result is significant Duncan’s Multiple Range test was used to assess the significance of inter-group differences. If significance is the result of the Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test.
Dams or litters were excluded from the data evaluation in cases of:
- Non pregnant females
- Body weight, body weight gain and food consumption of dams with complete intrauterine death of conceptuses (partial exclusion)
Although these animals were excluded from the data evaluation the Study Report contains all data of these animals, too.
A male/female fetus was considered as retarded in body weight, when its weight was below the average minus twofold standard deviation of the control male/female fetuses.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related clinical signs observed in the females. From the fourth treatment day onwards soft faeces was recorded for all cages including control which was attributed to the vehicle.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no significant differences in the body weight of the females. Minor but statistically significant increases were observed in the body weight gain if calculated for gestational days 5 to 8 in all treatment related groups and a lower body weight gain between gestation days 8 and 11 in the high dose group (p<0.05). The difference of 2.7 g or less compared to the control group were considered as biologically not relevant. The corrected body weight and corrected body weight gain were similar in all groups.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Slight but statistically significant increases of the food consumption were observed between gestation days 5 and 17 in the 300 mg/kg bw/day group as well as a minimal but statistically significant reduction in the low dose before the treatment period. These differences were considered as unrelated to the treatment.

Food efficiency:

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:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Statistical significance was observed in the values of early embryonic death, post-implantation loss in the 100 and 1000 mg/kg bw/day groups as well as total intrauterine mortality in the 1000 mg/kg bw/day group; however the values observed were lower compared to the values found in the control group.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

Disposition of females
The number of sperm positive females was 27 both in the control and 1000 mg/kg bw/day group as well as 23 both in the 100 and 300 mg/kg bw/day groups. There were five females in the control without any implantation (corpora lutea was present in one of these animals). At 100 mg/kg bw/day there were four females with no implantations (two of them had corpora lutea) and two with 3 or less implantations. One non-pregnant female and three non-pregnant females (no implantation and no corpora lutea) were found in the 300 and 1000 mg/kg bw/day dose groups, respectively. These females were excluded from the evaluation. In total, on gestation day 20 there were 22, 17, 22 and 24 evaluated litters each in the control, 100, 300 and 1000 mg/kg bw/day groups respectively.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No adverse effects observed.

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

not examined

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

The sex distribution of fetuses in the high dose group was statistically significantly different compared to the control; however this was without any biological relevance considering that the ratio was closer to 50 - 50 % than in the control group.

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The number of the affected litters was 2, 0, 1 and 1 in the control, 100, 300 and 1000 mg/kg bw/day group respectively.
One fetus in the mid dose had multiple malformations on the head as follows:
absent mandible; fused oral orifice; malformed maxilla and nasal orifice, anophthalmia (right) open eye, blister-like (left); one nasal orifice, microcephaly, malpositioned pinna (lower).
According to the experience with this species in this laboratory, multiple malformations of the head occurs sporadically without a relationship to the treatment. Considering also that no other fetuses were found in the test item treated groups with similar malformations this was judged as incidental.
One fetus was found in the 1000 mg/kg bw/day group with umbilical hernia. Considering that one control fetus had the same malformation this was judged to be unrelated to the test item.
In addition one fetus of the control group had a malrotated forelimb.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The number of the affected litters was 2, 1, 2 and 1 in the control, 100, 300 and 1000 mg/kg bw/day group respectively.
A misshapened xyphoid process of sternum in the control group, a split xyphoid process in the mid dose, split sternebra (in cartilage) one each in the control and high dose group, fused ribs one each in the low and mid dose group, hemicentric thoracic centrum (with or without a slightly dumb-bell shaped cartilage) one each in the mid and high dose group were found as malformations during skeletal examination.
These abnormalities occurred sporadically with low incidence or/and without a dose response. Furthermore most of these changes occur in control fetuses without a relationship to the treatment according to the historical control data.Therefore these malformations were judged to be unrelated to an effect of the test item.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The number of the affected litters was 1, 0, 2 and 0 in the control, 100, 300 and 1000 mg/kg bw/day group, respectively.
Microphthalmia was found in one fetus in the mid dose. The fetus with multiple head malformations at external examination in the mid dose was allocated to visceral examination which revealed malformed nasal cavity and nasal coanches, absent nasal septum, absent eyelid, anophthalmia(left), exophthalmia (right) and thin cerebral hemisphere. In the same litter enlarged perimeningeal space and hypoplastic cerebral hemisphere on the right side were recorded for another fetus. One control fetus had dilated lateral ventricles. Microphthalmia and multiple head malformations occur sporadically with low incidence in control fetuses according to the experience of this laboratory which is in line with the historical control data. Considering that only two litters were affected in the mid dose and none in the other test item treated groups besides the control group, these malformations were judged as incidental.

Details on embryotoxic / teratogenic effects:

External Variations
Clotted blood around the margin of the placenta was recorded for one fetus in the mid dose group and four fetuses in one litter in the 1000 mg/kg bw/day dose group. Based on the low litter incidence and that these fetuses had no external and visceral abnormalities or skeletal malformations this was considered to be not adverse. In the amnion of one control fetus blood was observed unrelated to the treatment.

Visceral variations
Hydroureter (distributed equally in the groups), hydroureter with dilated renal pelvis (one fetus in the high dose group) and slight testis dystrophy, unilateral (one fetus in the mid dose) were evaluated as variations. Considering the low incidences and/or the lack of dose response these variations were not attributed to the treatment.

Skeletal variations
Incompletely, not ossified or retarded skull bones, unossified hyoid bone, bipartite supra occipital, slightly hypoplastic mandible, incompletely ossified sternum, bipartite sternebra, cervical rib, wavy ribs, dumb-bell shaped, bipartite, asymmetrically ossified vertebral centra or slightly dumb-bell shaped cartilage, incompletely or not ossified sacral arches or centra, incompletely or not ossified pubic or ischii, asymmetric pelvic articulation of vertebral arches, slightly bent radius and ulna and asymmetric or incomplete ossification of metacarpal and metatarsal were evaluated as variations during the skeletal examination.
There was a statistical significant increase (p<0.01 fetal incidence and p<0.05 litter incidence) in the occurrence of wavy ribs at 1000 mg/kg bw/day. This variation was considered as non- adverse, becauseaccording to international scientific publications wavy ribs are reversible variations since “normalization of the neonate's homeostasis leads to enhanced ossification and concomitant bone repair until weaning” (Kast Exp Toxicol Pathol. 1994 Aug; 46(3):203-10.).
There was an increase seen in the incomplete ossification of skull bones however this was statistically not significant and considered as non- adverse. Based on scientific literature both variations are reversible. This assumption is supported by Carney (Birth Defects Res B Dev Reprod Toxicol. 2007 Dec; 80(6):473-96) who stated that both minor delays in ossification and wavy ribs seem to be readily repairable via postnatal skeletal remodeling and are not mechanistically linked to malformation.
There were no significant differences in the incidence of the other variations.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

Oral treatment of pregnant Hsd. Han: Wistar rats from gestation day 5 up to day 19 (the day before Caesarean section) with Incozol 4 at dose levels of 100, 300 and 1000 mg/kg bw/day did not cause death, clinical signs and necropsy findings and did not influence the food intake and body weight development of the maternal animals. Incozol 4 did not reveal any adverse effect on the pre- and postimplantation loss, number of implantation, sex distribution, body weight, placental weight, external, visceral and skeletal development of the fetuses. Lower placental weight and increased incidence of wavy ribs at 1000 mg/kg bw/day was considered as not adverse. Thus, a NOAEL of 1000 mg/kg bw/day was determined for maternal and developmental toxicity.

Executive summary:

Incozol 4 was examined for its possible prenatal developmental toxicity in a study according to OECD guideline 414. Groups of 23, 23 and 27 sperm-positive female Hsd. Han: Wistar rats were treated with Incozol 4 by oral administration daily at three dose levels of 100, 300 and 1000 mg/kg bw/day respectively from day 5 up to and including day 19 post coitum. A control group of 27 sperm positive females was included and the animals were given the vehicle PEG 400. The treatment volume was 5 mL/kg bw.

A sufficient stability and homogeneity in the chosen vehicle was verified over the range of relevant concentrations at the appropriate frequency of preparation. Incozol 4 in PEG 400 was stable at room temperature for at least four hours and for one day in the refrigerator (5 ± 3 °C) at the concentrations of 10 and 200 mg/mL. Analytical control of dosing solutions was performed during the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 93 and 110 % of nominal concentrations at both analytical occasions confirming proper dosing.

During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day, when sperm was detected in the vaginal smear, was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method.

After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.

In total, on gestation day 20 there were 22, 17, 22 and 24 evaluated litters in the control, 100, 300 and 1000 mg/kg bw/day group respectively.

None of the females died before scheduled necropsy and there were no test item related clinical signs recorded. No treatment related necropsy findings were observed. No treatment related food reduction or body weight alterations were determined in the dams. Number of implantations, intrauterine mortality and sex distribution of the fetuses were not influenced by the treatment.

There were no test item related adverse effects on the fetal weight, external and visceral development of fetuses. There were no test item related malformations found. The number of litters with malformations was 3, 1, 4 and 2 in the control, 100, 300 and 1000 mg/kg bw/day groups respectively.

Statistically significant increase of incidence of wavy ribs in the 1000 mg/kg bw/day group was observed, but the value was within the historical control range. Based on scientific literature this variation is reversible hence not an indicator of an adverse effect. Relative placental weight (placenta mg: fetal g) was statistically significantly lower at 100, 300 (within the historical control range) and 1000 as well as the absolute and relative placental weight at 1000 mg/kg bw/day. Considering that there was no impact on the body weight development of the fetuses and no adverse changes were observed, the reduced placenta weight was regarded as non-adverse.

Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:

NOAEL (maternal toxicity): 1000 mg/kg bw/day

NOAEL (developmental toxicity including teratogenicity): 1000 mg/kg bw/day