Hexane-1,3,6-tricarbonitrile

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022.6.21-2023.01.16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Hexane-1,3,6-tricarbonitrile

EC Number:

217-199-7

EC Name:

Hexane-1,3,6-tricarbonitrile

Cas Number:

1772-25-4

Molecular formula:

C9H11N3

IUPAC Name:

hexane-1,3,6-tricarbonitrile

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: 11-12weeks
- Weight at study initiation:
males: 310 - 400 g
(mean: 368.04 g, ± 20% = 294.43 – 441.65 g)
females: 180 - 236 g
(mean: 205.43 g, ± 20% = 164.34 – 246.51 g)

- Fasting period before study: yes
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period:7days under laboratory condition

ENVIRONMENTAL CONDITIONS
- Full barrier in an air-conditioned room
- Temperature: 22  3 °C
- Relative humidity: 40–70%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

castor oil

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

Mating was performed using a ratio of 1:2 (male to female). Females were paired for cohabitation in batches in order to control the number of animals for terminal sacrifice on a particular day. At the subsequent mornings, the vaginal smear of the females was checked to confirm the pregnancy. The day on which sperm is observed in the vaginal smear is considered as “GD 0”. Mated females were assigned in an unbiased manner to the control and treatment groups ensuring that group mean body weights are comparable with each other. Each animal was assigned a unique identification number. After getting 92 sperm-positive females, the remaining females and males were discarded without any observation.

Duration of treatment / exposure:

14days, gestation day (GD) 5 to 19

Frequency of treatment:

The animals were treated with the test item or vehicle on 7 days per week from GD 5 to GD 19.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

250 mg/kg bw/day (nominal)

No. of animals per sex per dose:

138 animals (46 males and 92 females) were included in the study.

Control animals:

yes

Examinations

Ovaries and uterine content:

On GD 20, sperm-positive (presumed pregnant) females were subjected to a caesarean section after sacrificing the animals using anaesthesia (ketamine/xylazin).
Females showing signs of abortion or premature delivery prior to scheduled termination were sacrificed and subjected to a thorough macroscopic examination.
At the time of termination or death during the study, each dam (presumed pregnant female) was examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy. Any macroscopic findings were preserved in 4% neutral-buffered formaldehyde.
Immediately after the termination or as soon as possible after death, the uteri were removed and the pregnancy status of the dams was confirmed. Uteri that appeared non-gravid were further examined by staining with 10% ammonium sulphide solution to confirm the non-pregnant status.
Each gravid uterus with cervix was weighed. However, the gravid uterus obtained from dead animals was not weighed.
Thyroid/parathyroid glands from all dams were preserved in 4% neutral-buffered formaldehyde. The weight of thyroid/parathyroid glands was measured after 24 hours fixation.
The number of corpora lutea was counted for pregnant animals. The uterine contents were examined for embryonic or foetal deaths and the number of viable foetuses. The degree of resorption (late and early) was confirmed in order to help estimate the relative time of death of the conceptus. The position and number of foetuses in each uterine horn was recorded.
Males were sacrificed without any observation at any time after the completion of the mating or were used for other studies.

Fetal examinations:

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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Description (incidence):

One HD group dam (No. 90, non-pregnant) was sacrificed in moribund condition on GD 14 for the animal welfare reasons. Reduced spontaneous activity (moderate), piloerection (moderate and moving the bedding material were observed on GD 14 post dose and It did not show any clinical signs prior to the day of dosing until GD 13. Slight reduction in mean body weight (8.18%) and lowered food consumption were observed on GD14 when compared to day 1. At necropsy, stomach with abnormally coloured (black, spotted at cardiac area, several) were observed. No histopathology of this animal was performed and hence the cause of the death could not be determined microscopically. However, based on the clinical end points observed in the other HD group and lower dose groups dams, it is not considered to be a test item related effects and incidental.

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:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

no effects observed

Description (incidence and severity):

In all terminally sacrificed females, no statistically significant or test item related effects was observed on group mean T3, T4 and TSH hormone levels and values were comparable with the control.
Statistical analysis of post-fixed thyroid/parathyroid weights from all dams revealed no statistically significant or toxicologically relevant effect on the absolute and relative (to body weight) thyroid/parathyroid weights of the dose groups when compared to the control.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

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)

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No adverse effect observed

Maternal abnormalities

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:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect observed

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Treatment related:

no

Applicant's summary and conclusion

Conclusions:

On the basis of this prenatal developmental toxicity study with Hexane-1,3,6-tricarbonitrile pregnant female Wistar rats with dose levels of 50, 150, and 250 mg/kg body weight day the following conclusions can be made:
No test item-related mortality and clinical signs of toxicity were observed in any of the treatment groups during the study period.
No test item-related effects on mean body weight and mean food consumption were observed in any of the treatment groups.
No test item-related effects were observed on prenatal parameters including terminal body weight, adjusted maternal weight (carcass weight), uterine weight, number of corpora lutea, implantation sites, early and late resorptions, percent pre and post implantation loss, number of live foetuses, anogenital distance (AGD), foetal weight, number of male and female foetuses, sex ratios and testicular descent in treatment groups when compared to the control.
No statistically significant or toxicologically relevant effect was observed on group mean T3, T4 and TSH hormone levels and thyroid/parathyroid weights from all dams. No test item-related findings at histopathological evaluation of the thyroid gland in any of the treatment groups.
No test item-related effects on gross pathology lesions were observed in any of the treatment groups.
No test item related and toxicologically relevant external, visceral, craniofacial and skeletal findings were observed in the foetuses of all treatment groups when compared to control group.
The NOAELs for maternal and foetal toxicity of Hexane-1,3,6-tricarbonitrile were considered to be 250 mg/kg body weight/day.

Executive summary:

The objective of this study was to assess the possible adverse effects on pregnant females and embryo-foetal development which could arise from repeated exposure of Hexane-1,3,6-tricarbonitrile via oral administration to female rats from gestation day (GD) 5 to 19.

Nulliparous and non-pregnant females were mated with males (2:1 ratio) and divided into four groups based on their body weights on the day of sperm-positive vaginal smears (GD 0). The groups comprised 23 female Wistar rats each. Animals of the control group were handled identically as the dose groups but received the vehicle instead of test item formulation.

The following doses were evaluated:

Control:            0 mg/kg body weight/day

Low Dose:      50 mg/kg body weight/day

Medium Dose: 150 mg/kg body weight/day

High Dose:   250 mg/kg body weight/day

The test item formulation was prepared at least every 10 days (stability time frame as given by Eurofins Agroscience Services Study No. S22-03900). The test item was diluted in 2% Kolliphor in aqua ad injectionem and administered daily from GD 5 to GD 19 to presumed pregnant female animals (dams). Dose volumes were adjusted individually based on weekly body weight measurement.

During the period of administration, the animals were observed precisely each day for signs of toxicity and mortality. Body weight and food consumption were measured on GD 0, 5, 8, 11, 14, 17 and 20.

Animals which was moribund scarified during the study were examined macroscopically. All surviving female animals were sacrificed on the respective GD 20. Following the gross necropsy, the uteri and ovaries were removed, weighed and examined for number of implantations, resorptions (early and late), live and dead foetuses. Foetuses were identified using numbered plates, sexed and weighed. All foetuses were observed for external abnormalities, half of the foetuses for visceral and craniofacial abnormalities and the remaining half of the litter was observed for skeletal abnormalities.

The uteri of the non-pregnant females were processed with 10 % ammonium sulphide solution and checked for the early embryonic deaths.

Thyroid/parathyroid glands from all dams were preserved. The weight of thyroid/parathyroid glands was measured after fixation. Blood samples were assessed for serum levels for thyroid hormones.

A histopathological evaluation of the preserved thyroid/parathyroid glands was performed.

 

Maternal Findings

No test item-related mortality was observed during the treatment period. One mortality occurred in HD female (no.90) on GD 14 which was assumed to be incidental. All animals survived until the end of the study.

There was no test item-related clinical signs of systemic toxicity observed in the females of any treatment groups. Salivation and moving the bedding materials were observed in the treated groups were considered to be local reaction to the treatment with test item. None of the females showed signs of abortion or premature delivery prior to the scheduled necropsy.

The mean body weight and mean body weight gain remained unaffected and increased with progress of the study in all treatment and control groups. No test item-related effect was observed in food consumption in all the treatment groups when compared to control.

Successful mating resulted in 21/23 pregnancies in the LD group, 19/23 in the MD group and 21/23 in the HD group compared to 21/23 pregnancies in the control group.

No test item-related effects of toxicological relevance were noted for any prenatal parameters including terminal body weight, adjusted maternal weight (carcass weight), uterine weight, number of corpora lutea, implantation sites, early and late resorptions, pre- and post-implantation loss, number of live foetuses, number of male and female foetuses and sex ratios in treatment groups when compared to the control group. No dead foetuses were noted in any of the groups.

No statistically significant or test item-related effects were observed on group mean T3, T4 and TSH hormone levels and values were comparable to the control group.

Statistical analysis of post-fixed thyroid/parathyroid weights from all dams revealed no statistically significant or toxicologically relevant effect on the absolute and relative (to body weight) thyroid/parathyroid weights of the dose groups when compared to the control.

No test item related macroscopic observation were observed in any of the treatment groups.

There were no test item-related findings at histopathological evaluation of the thyroid gland in any of the treated groups observed.

Foetal Findings

In male and female foetuses, body weight in treatment groups remained unaffected and no statistical significance was observed in any of the treatment groups when compared to the control group.

No test item related changes were observed in anogenital distance (AGD) were observed in the treated groups. All male foetuses were checked for indication of incomplete testicular descent/ cryptorchidism and evaluation revealed completion of testicular descent (abdominal) in all male foetuses from all groups.

No test item-related and statistically significant changes in mean foetal weight, male and female foetal weight on a per litter basis (group mean of individual litter mean) were observed in any of the treatment groups when compared to the control group.

There was no test item-related external abnormalities observed in any of the treated groups.

Internal observation of the foetal viscera by free hand microdissection technique revealed a range of visceral findings in all groups including the control group. Visceral findings observed in the dose groups were at frequencies generally comparable to or in some cases slightly higher or lower in frequency compared to controls. As the observed findings were minor variations and/or lacked dose dependency and consistency, no toxicological significance can be attributed to these findings and they were considered to be spontaneous in nature. All litter incidences from dose groups were within the historical control data range and no statistically insignificant changes were observed in the treatment groups when compared with the control group.

Craniofacial examination by razor blade serial sectioning technique did not reveal any test item related findings when compared to control. All the findings were considered to be spontaneous in nature and not related to the treatment with the test item and were within the historical control range. Statistical analysis of the data revealed no statistical significance in any of the dose groups when compared to control group.

Skeletal examination and examination of cartilage of the Alizarin red stained foetuses revealed a range of findings in all groups including control.

Statistically significantly lower foetal incidences were observed at skull interparietal, incomplete ossification in MD group (17.52%) when compared to control (51.67%). Statistically significantly lower foetal incidences were observed at bent scapular spine in all treatment groups (0%) when compared to 2.67% in the control. Statistically significantly lower foetal incidences were observed at wavy ribs in MD group (3.43%) when compared to 26.49% in the control. Statistically significantly higher foetal incidences were observed at 5^th sternebra (incomplete ossification and unossified) in HD group (79.27%) when compared to 51.40% in the control. All these findings were observed without dose dependency or consistency; hence they are not considered to be test item-related.

 

Other skeletal findings were observed without achieving statistical significance or dose dependency. The observed incomplete ossification of some bones and other incidental skeletal findings in the treated groups were either marginally lower or higher than concurrent controls or within the historical control data range. Generally, delayed ossification is not regarded to persist post-natally and is not associated with long-term consequences on survival, general growth and development and, therefore, is not considered to be adverse [11], [12].

Dose Formulation Analysis

Formulation analysis for concentration verification was performed on collected samples during the study (first and last week of the study). Nominal concentrations were confirmed for all dose groups, as the measured concentrations did not differ from nominal concentrations by more than 15%.

 

On the basis of this prenatal developmental toxicity study with Hexane-1,3,6-tricarbonitrile pregnant female Wistar rats with dose levels of 50, 150, and 250 mg/kg body weight day the following conclusions can be made:

No test item-related mortality and clinical signs of toxicity were observed in any of the treatment groups during the study period.

No test item-related effects on mean body weight and mean food consumption were observed in any of the treatment groups.

No test item-related effects were observed on prenatal parameters including terminal body weight, adjusted maternal weight (carcass weight), uterine weight, number of corpora lutea, implantation sites, early and late resorptions, percent pre and post implantation loss, number of live foetuses, anogenital distance (AGD), foetal weight, number of male and female foetuses, sex ratios and testicular descent in treatment groups when compared to the control.

No statistically significant or toxicologically relevant effect was observed on group mean T3, T4 and TSH hormone levels and thyroid/parathyroid weights from all dams. No test item-related findings at histopathological evaluation of the thyroid gland in any of the treatment groups.

No test item-related effects on gross pathology lesions were observed in any of the treatment groups.

No test item related and toxicologically relevant external, visceral, craniofacial and skeletal findings were observed in the foetuses of all treatment groups when compared to control group.

The NOAELs for maternal and foetal toxicity of Hexane-1,3,6-tricarbonitrile were considered to be 250 mg/kg body weight/day.