N-[3-(trimethoxysilyl)propyl]butylamine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Feb 2021 - xx

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-[3-(trimethoxysilyl)propyl]butylamine

EC Number:

250-437-8

EC Name:

N-[3-(trimethoxysilyl)propyl]butylamine

Cas Number:

31024-56-3

Molecular formula:

C10H25NO3Si

IUPAC Name:

butyl[3-(trimethoxysilyl)propyl]amine

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI (Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at arrival test facility: females: 11-12 weeks old; males: 12-13 weeks old
- Weight at study initiation: females: 173 – 248 g (mean: 216.21 g, ± 20% = 172.97 – 259.45 g); males: 312 -366 g (mean: 340.60 g, ± 20% = 272.48 – 408.73 g)
- Fasting period before study: no
- Housing: individually in IVC cages (type III H, polysulphone cages) on Altromin saw fibre bedding (except during the pre-mating period when females were kept in groups of two animals and during mating period when two females were paired with one male); the pregnant females were provided with nesting material towards the end of the pregnancy (e.g. at GD 18)
- Diet: Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water: tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals), ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

dried and de-acidified

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved in dried and de-acidified corn oil. Corn oil was filtered through a mixture of activated silica gel 60 and aluminium oxide (1:1, volume/volume), which had been filled into a glass chromatography column to three quarters of its height. For filtering, a vacuum of 75 mbar was applied. The dried and de-acidified vehicle was overlaid with argon and stored until usage.
The test item was weighed into a tared glass vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item. The formulation was alternately vortexed and/or stirred until visual homogeneity was achieved.
After homogenization the formulation was overlaid with argon to prevent instability caused by repeated contact of the test item formulation with air.
Based on the results of stability testing, the test item formulations were prepared freshly each day of administration. The prepared formulation was stored protected from light and at room temperature.


VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected as suggested by the sponsor based on the test item’s characteristics and testing guideline.
- Concentration in vehicle: 12.5, 30 and 75 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
- Lot/batch no.: MKCM3364

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before the beginning of the treatment period, formulation samples of a concentration range of 50-250 g/mL were prepared and analysed in order to obtain knowledge about the stability and homogeneity of the test item in the selected vehicle as part of a separate GLP study. The formulations (50, 125 and 250 mg/mL) were stable for 2 hours, 4 hours and for 2 days at room temperature, and for 2 days at 2 to 8 °C. An additional validation was conducted prior to the start of the study to yield formulation stability and homogeneity data up to the lower concentration of 12.5 mg/mL.
Pre-start homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups.
As the test item formulation was shown to be homogenous (after 30 min without stirring), samples were not collected during the study for the investigation of homogeneity and samples were only taken for substance concentration verification in the first and last week of the study for all doses (8 samples in total).
Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 3 mL). The A-samples were analysed and until then stored under appropriate conditions based on available stability data. The B-samples were retained at below -15 °C at the test facility and discarded after completion of the final study report.
Nominal concentrations were confirmed for all dose groups, as measured mean concentrations were within the acceptance criterion of 10%.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:2
- Length of cohabitation: Females were paired for cohabitation in batches in order to control the number of animals for terminal sacrifice on a particular day. After getting 92 sperm positive females, the remaining females and males were discarded without any observations.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

Day 5-19 of gestation

Frequency of treatment:

daily, 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

low dose (LD)

Doses / concentrations 2

Dose / conc.:

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

Remarks:

medium dose (MD)

Doses / concentrations 3

Dose / conc.:

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

Remarks:

high dose (HD)

No. of animals per sex per dose:

23 mated females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In the 14-day repeated dose range finding study (BSL Munich Study No. 192940, non GLP), pregnant female Wistar rats were treated with the test item at doses of 300 (LD), 600 (MD) and 1000 (HD) mg/kg bw/day.
Mortality was observed in all dose groups (LD and MD 1/8 female each and HD 3/8 females). These females had to be euthanized in a moribund condition showing vocalization and severe clinical signs of the respiratory/pulmonary tract. Additionally, they showed macroscopic findings in the gastrointestinal tract (gas-filled and abnormally colored stomach, ileum, jejunum and caecum), which were assumed to be a local reaction to the test item.
Test item-related clinical signs were observed in all treatment groups, but more severe and more frequently in higher dose groups. In all females that were sacrificed as scheduled on GD 20, no gross pathological findings were seen. No test item-related effects were observed in terms of body weight, body weight gain, food consumption, prenatal/litter data and no signs of foetal toxicity were seen up to the highest dose level of 1000 mg/kg bw/day when compared to the control group.
Taking into account the results of the 14-day repeated dose range finding study, a local, but marked toxicological effect of the test item on the respiratory/pulmonary and gastrointestinal tracts of the pregnant females, which lead to mortality in all dose groups, was likely.
The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence of dose levels were selected with an aim to demonstrate any dose-related response and to identify a NOAEL.

- Rationale for animal assignment: Before the first administration all animals to be used for the study were weighed and assigned to the experimental groups with the aim of achieving minimum variation in body weight throughout the groups of males and females, respectively (randomisation was performed with IDBS Workbook 10.5.0 software).
- Fasting period before blood sampling for (rat) dam thyroid hormones: no
- Time of day for (rat) dam blood sampling: Thyroid hormone levels from samples from all dams were assessed at the end of treatment prior to or as part of the sacrifice of the animals.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once a day, perferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
- Cage side observations checked: spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size, changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to the start of the mating a detailed clinical observation was made outside the home cage.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed once before initiation of pairing to ensure that the body weights were within ± 20 % variation. The sperm positive females were weighed on GD 0, 5, 8, 11, 14, 17 and 20. Males were not be weighed in this study except once before initiation of pairing.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption of sperm-positive females was determined for the following intervals: GD 0-5, 5-8, 8-11, 11-14, 14-17 and 17-20. Food consumption was not measured for males during the entire study or for both males and females during the mating period.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined:
Each dam 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.
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.
Special attention was given to the pulmonary / respiratory and gastrointestinal tracts in order to assess possible treatment-related effects. Therefore, all organs listed in Table 1 from all dams underwent a detailed macroscopic observation and were preserved in 4% neutral-buffered formaldehyde for possible histopathological examination:
- The oesophagus was opened by a longitudinal cut with a scissor (without rinsing).
- The stomach was rinsed after opening with a cut and the mucosa was inspected.
- The small intestine was separated from the large intestine by a cut at the entry of the ileum into the caecum. Thereafter, the small intestine was cut into 3 pieces whereby the proximal end was marked by a shaped cut, and the intestine was gently rinsed with formalin (if the intestine was too full, small cuts allowed the outflow of ingests and rinsing fluid).
- The caecum was opened by a longitudinal cut and rinsed. Colon and rectum was also rinsed.
A histopathological evaluation was carried out on the preserved thyroid/parathyroid glands from all dams of all dose groups sacrificed at the end of the treatment period.

OTHER: Thyroid hormones levels from samples from all dams were assessed at the end of the treatment prior to or as part of the sacrifice of the animals. At termination, blood samples were collected from the defined site in serum separator tubes and obtained serum was stored under appropriate conditions. Serum samples were assessed for serum levels for thyroid hormones (T3, T4, TSH) using ELISA.

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

Blood sampling:

- Plasma: No
- Serum: Yes (for evaluation of thyroid hormones)

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

A statistical assessment of the results of the body weight and food consumption was performed by comparing values of dosed animals with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, thyroid hormones and foetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. The statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.3.4 software (p<0.05 is considered as statistically significant).

Historical control data:

Results were compared with historical control data.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs observed occasionally during the treatment period of the study included, hairless area on skin and fur in one control animal (no. 16) on its abdomen area on GDs 18-20.
One LD group animal (no. 34) showed moderate wasp waist on GDs 6-7 and slight piloerection on GDs 5-7.
Two MD group animals (nos. 51, 57) showed abnormal breathing on GDs 13-14 and 19-20, respectively. Slight piloerection was observed in MD animal no. 55 on GD 17. Animal no. 60 showed abnormal breathing on GD 13 and moderately reduced spontaneous activity, abnormal breathing, nasal discharge on GDs 15-16 as well as moving the bedding on GDs 15-17. Animal no. 64 showed right side chromodacryorrhea on GD 6-18, slight piloerection on GDs 11-15, lacrimation and eye swelling on GD 20 of the right eye. Animal no. 65 showed slight piloerection on GD 15 and cough/sneezing on GDs 13-14.
HD group animals (nos. 74, 76, 78) showed abnormal breathing on GDs 8, 16-19 and 9-11, respectively. Slight piloerection was observed in animal nos. 78, 87, 89, 90 on GDs 9-11, 16-19, 15 and 14-17, respectively. Animal no. 70 showed slight wasp waist on GDs 11-12, sight piloerection on GDs 13-14, moderate wasp waist om GDs 13-1 and GDs 19-20, and moderate piloerection on GDs 19-20. Animal no. 92 showed slight piloerection on GDs 13-14, moderate piloerection on GDs 15-20, severe increased salivation on GDs 15-16, abnormal breathing on GDs 16-20, and moderately increased salivation on GD 17.
Salivation and moving the bedding materials are considered to be signs of a local reaction to the test item. These clinical signs were observed on a few days of the treatment period, immediately after dosing. Abnormal breathing, piloerection, reduced spontaneous activity, nasal discharge and lacrimation are considered to be test item-related and caused by aspiration/regurgitation of test item during the treatment period of this study.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

LD group animals (no. 34, 44) were euthanized on GDs 13 and 11 respectively, due to clinical signs of severe/slight piloerection, abnormal breathing and salivation in animal no. 34, and severe/moderate piloerection, abnormal breathing and closed eyelids of both eyes in animal no. 44.
MD animal no. 69 was found dead on GD 14. It showed clinical signs of severe piloerection, abnormal breathing, slight salivations and moderately reduced spontaneous activity prior to death.
HD group animals (no. 81, 84 and 91) were found dead on GDs 18, 15 and 11 respectively, after dose application and those animals did not show any clinical signs prior to death.
All euthanized/found dead rats were found to be pregnant at necropsy.
Based on the gross lesions in the thoracic cavity, lungs and stomach, and clinical signs in the moribund or dead animals of treated groups, the cause of the morbidity or death is assumed to be due to aspiration/regurgitation of the test item during the treatment period.
No mortality occurred in the control group during the treatment period.
All other females survived until the date of their scheduled sacrifice.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean body weight and mean body weight gain increased with the progress of the study in the control and all the treated groups throughout the study period.
A slight but statistical significant effect was observed in the MD group on GDs 14 (5.75% above control) and 17 (6.66% above control). There was a slight increase or decrease in mean body weight gain observed on GDs 5-8 (40.98% above control in the LD group and 11.48% and 6.56% below control in the MD and HD groups, respectively), and on GDs 8-11 (13.69% below control in the LD group). Increased mean body weight gain was observed in all dose groups on GDs 11-14 (LD: 72.97%, MD: 63.87% and HD: 12.61%). Overall, there was slight increase in mean body weight gain on GDs 0-20 observed in the MD group. As there was no dose-dependency or consistency in these alterations, they are not considered to be test item-related. Hence, no test item-related effects on mean body weight or mean body weight gain were observed in any of the treated groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean food consumption observed during GDs 5-20 in the LD, MD and HD groups were comparable to control. No statistical significance was observed in measured mean food consumptions. No test item-related effect on mean food consumption was observed in the treated groups.

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 specified

Description (incidence and severity):

The following ED-related parameters were investigated in the study: T3, T4 and TSH level dams, anogenital distance, genital abnormalities (testicular descent/cryptorchidism), thyroid histopathology dams, thyroid weight dams, gravid uterus weight, litter size, litter/foetus weight, number of implantations, corpora lutea, number of embryonic or fetal deaths and viable foetuses, post-implantation loss, pre-implantation loss, presence of anomalies (external, visceral, skeletal) and sex ratio. For details, please refer to the respective result fields and the endpoint summary.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Statistical analysis of post-fixed thyroid/parathyroid weights from all dams revealed, slight but statistically significant differences in the absolute and relative (to body weight) thyroid/parathyroid weights in LD group (absolute, 25.38% above control) and absolute and relative (to body weight) in HD groups (31.67% and 27.50% respectively, above control) when compared to the control. At the histopathology evaluation, there were no histology changes in the thyroid and parathyroid glands that could be related to the treatment with the test item; hence the sight the change in statistical significance is not considered to be treatment related.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

LD groups: Animal no. 36 had fluid filled thoracic cavity with creamy white content. Animal no. 40 had white fluid filled thoracic cavity, white colored lungs and small thymus. Animal no. 44 had thoracic cavity with oily fluid filled, dark colored lungs with abnormal consistency, adhesion at thorax, dark red and enlarged (both side) adrenal glands, animal no. 36 had dark red ovaries and animal no. 29 had red colored duodenum at necropsy.
MD groups: Animal no. 69 had reddish colored lungs, lungs with failure to collapse, gas filled stomach, dark, oily fluid filled vagina and reddish enlarged adrenal glands. Animal no. 55 had dark red ovaries. Animal no. 50 had cyst in the thymus and animal no. 62 had spotted red in thymus.
HD groups: Animal no. 91 had thoracic cavity with fluid filled content, dark red colored lungs. Animal 81 and 84 had dark lungs and abnormal colored thymus. Animal no. 81 had lungs with failure to collapse. Animal no. 73 had cyst in the thymus.
Based on the gross lesions in the thoracic cavity, lungs and stomach and clinical signs in the treated groups of moribund or dead animals; the cause of the morbidity or death are assumed to be due to aspiration/regurgitation of test item during the treatment period.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test item-related histopathological findings were observed in thyroid glands and parathyroid of all females.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

In all terminally sacrificed females, no statistically significant or toxicologically relevant effects were observed on group mean T3, T4 and TSH hormone levels and values were comparable to the control.

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

None of the females showed any signs of abortion or premature delivery prior to their scheduled sacrifice.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No test item-related effects were observed on percent pre- and post-implantation loss.

Total litter losses by resorption:

not specified

Early or late resorptions:

no effects observed

Description (incidence and severity):

No test item-related effects were observed on early and late resorptions.

Dead fetuses:

no effects observed

Description (incidence and severity):

No dead foetuses were noted in any of the groups.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Successful mating resulted in 19/23 pregnancies in the LD group, 20/23 in the MD group and 21/23 in the HD group compared to 18/23 pregnancies in the control group. All moribund or dead animals (2 LD, 1 MD, and 3 HD animals) were found to be pregnant at necropsy.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related effects of toxicological relevance were noted for any prenatal parameter, including terminal body weight, adjusted maternal weight (carcass weight), uterine weight, number of corpora lutea and implantation sites in the treatment groups when compated to the control group. A slight but statistically significant increase in carcass weight was observed in the MD group (4.71% above control). All these values are within the historical control range of this strain.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Based on:

test mat.

Basis for effect level:

clinical signs

gross pathology

mortality

Remarks on result:

other: NOAEL for maternal toxicity could not be established due to the equivocal nature of the adverse effects seen in the moribund and dead animals in all of the test-item treated groups

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean male and female foetal weights observed on an individual basis (sum of weight of all foetuses in group divided by total number of foetuses in respective group) in the LD, MD and HD groups were comparable to control and no relation to test item or statistical significance were observed in any of the groups.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No test-item related effects of toxicological relevance were noted on the number of live foetuses in the treatment groups when compared to the control group.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No test-item related effects were noted in the number of male and female foetuses and sex ratios in the treatment groups when compared to the control group.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

No test item-related and statistically significant effects 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 treated groups when compared to the control group.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

In males and female foetuses, the absolute and relative anogenital distance (AGD) in treatment groups remained unaffected when compared to the control group.
All male foetuses were checked for indications of incomplete testicular descent/ cryptorchidism and evaluation revealed the completion of testicular descent (abdominal) in all male foetuses from all groups.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No test item-related external abnormalities were observed in any of the treated groups.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal examination and examination of cartilage of the Alizarin red stained foetuses revealed a range of findings in all groups including control.
As usual for foetuses at the examined stage of GD 20, incomplete ossification was seen in several bones of litters from all treated groups as well as the control. Mostly, bones of the skull, sternum, paws, limbs and vertebra were affected by variations in the status of expected ossification, described in terms of incomplete ossification, irregular ossification, unossified or increased ossification throughout all experimental groups. However, there was no dose dependency and no trend towards a treatment-related incomplete ossification of bones. Statistical analysis did not reveal any significance in the treated groups when compared to control.
Bones showing a higher litter incidence of incomplete ossification in the HD group compared to the control group, which did not reach statistical significance were skull inter parietal bone (83.3% compared to 77.8%), skull zygomatic arch (L) (16.7% compared to 5.6%), skull basisphenoid (22.2% compared to 16.7%) and 6th sternebra (100% compared to 94.4%). Bones showing higher litter incidence of being unossified in the HD group compared to the control group, which did not achieve statistical significance were forelimb phalanx/(ges) (100% compared to 99.07%). Bones showing a higher or lower litter incidence in the HD group compared to the control group, which did not achieve statistical significance were skull supraoccipital, with small hole (R) (72.2% compared to 77.8%), irregular ossification of vertebra cervical arch (22.2% compared to 33.3%) and rudimentary 14th rib in the HD group (R) (27.8% compared to 66.7%), and wavy ribs in HD group (61.1% compared to 66.7%).
In general, wavy ribs are typically classified as transient and reversible post-natally and may thus be considered as variations but not malformations. Observed ossification-related findings were seen at lower or higher incidences without dose-dependency and thus were not considered as toxicologically relevant.
There were also slightly higher or lower litter incidences of skeletal findings in the LD and MD groups, but they did not show any statistical significance or dose-dependency.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No statistical significance was observed in any of the treated when compared to the control group. Slightly higher litter incidences of azygos vein (bilateral) were observed in the HD group (16.7%) when compared to the control (11.1%). Higher litter incidences for umbilical artery malpositioned were observed in the LD and MD groups (58.8% and 57.9% compared to 50.0% in the control). Higher litter incidences of liver, supernumerary lobe were observed in the MD and HD groups (26.38% and 38.9%, respectively, compared to 22.2% in the control group). Higher litter incidences of abdomen internal haemorrhage were observed (78.9% and 83.3% in the MD and HD groups, respectively, compared to 72.2% in the control). Slightly higher litter incidences of thorax internal haemorrhage were observed (11.8% and 10.5% in the LD and MD groups, respectively, compared to 0% in the control). Higher litter incidences of long thymus were observed (29.4%, 26.3% and 33.3% in the LD, MD and HD groups, respectively, compared to 22.2% in the control).
Visceral findings observed in the treated groups were at frequencies generally comparable to or, in some cases, slightly higher or lower in frequency compared to the control. As the observed findings were either minor variations and/or due to a lack of dose dependency and consistency, no toxicological significance could be attributed to these findings and they were considered to be spontaneous in nature. In addition, all these values were within the historical control range for this strain.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Craniofacial examination by razor blade serial sectioning technique revealed a finding of head, subcutaneous hematoma (general) in the LD group (5.9%) when compared to control group (0%) and a comparable finding in brain with red material in perimeningeal space in the HD group (11.1%) when compared to control group (11.1%). Midbrain, haematoma, subdural in LD and HD groups (5.9% and 5.6% respectively) were observed at higher incidences when compared to control (0%).
These findings were considered to be spontaneous in nature and not related to the treatment with the test item. Statistical analysis of the data revealed no statistical significance for any of these findings. In addition, all values were within the historical control range for this strain.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Remarks:

foetal toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

In an OECD 414 compliant study conducted according to GLP, morbid or dead animals were observed in all treated groups. Based on the gross lesions in the thoracic cavity, lungs and stomach and clinical signs in the moribund or dead animals of treated groups, the cause of the morbidity or death can be assumed to be due to aspiration/regurgitation of test item during the treatment period. Therefore, the NOAEL for maternal toxicity could not be established due to the equivocal nature of the adverse effects seen in the moribund and dead animals in all of the test-item treated groups. The NOAEL for foetal toxicity could be established at 300 mg/kg bw/day, the highest dose tested in this study.