Dimethoxydimethylsilane

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Jul - 17 Sep 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dimethoxydimethylsilane

EC Number:

214-189-4

EC Name:

Dimethoxydimethylsilane

Cas Number:

1112-39-6

Molecular formula:

C4H12O2Si

IUPAC Name:

dimethoxydimethylsilane

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: approx. 11-12 weeks old for females; between 13 weeks and not older than 24 weeks for males
- Weight at study initiation: males: 341 – 394 g; females: 211 – 284 g
- Fasting period before study: no
- Housing: individually housed in IVC cages 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; during the pre-mating period and after mating, males were housed in groups (up to 5 animals / cage) in type IV cages
- Diet: Altromin 1324 maintenance diet for rats and mice provided ad libitum
- Water: tap water, sulphur acidified to a pH of approximately 2.8 provided 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

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item. The formulation was vortexed and/or stirred until visual homogeneity was achieved. After homogenization the formulation was overlaid with argon to prevent chemical reactions caused by repeated contact of the test item formulation with air. The prepared formulation was stored at room temperature and protected from light. Formulations were kept under magnetic stirring during the daily administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was selected in consultation with the sponsor based on the test items characteristics. The test item was dissolved in dried and deacidified corn oil.
- Concentration in vehicle: 25, 75 and 250 mg/mL
- Amount of vehicle: 4 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle at Eurofins Munich as part of a separate GLP study (Eurofins Munich Study No. 188370).
Study prestart stability analysis was included on the samples from high-dose and low-dose group and the investigation was made for 0 h, 6 h (RT), 7 days (RT), 7 days (2 to 8 °C) and 7 days (-15 to -35 °C). All stability tests were passed as concentrations were found to be within the acceptance range of ≤±15%.
Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high- (HD) and low-dose (LD) groups.
As the test item was shown to be homogenous according to Eurofins Study No. 188370 (after 30 min without stirring), samples were not collected during the study for the investigation of homogeneity. Samples were taken for verification of the concentration in the first, second, third and last week of the study for all doses (24 samples in total).
The mean recoveries observed for the LD dose group was between 90.2% and 104.8% of the nominal value, between 94.2% and 101.8% for the MD dose group and between 87.1% and 103.0% of the nominal value for HD dose group. The mean recoveries observed in the LD, MD and HD groups were 97.8%, 97.9%, and 95.5% of the nominal concentration, respectively.
Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 15%. However, samples (LD: week 1 and 2 - first measurement; MD: week 1 and 2 - first measurement; HD: week 1 -repetition) were excluded from the overall mean calculation or evaluation because these samples did not meet the acceptance criterion (recoveries were < 85%).
Samples from week 1 were reanalysed (a- and b-aliquots) and the reanalysis confirmed the results of the first measurement. This indicated a technical error in the preparation of formulation samples of week 1 for LD and MD. Second batches of LD-, MD- and HD samples were additionally prepared in week 1 and similar results were shown for LD- and MD samples. Also in week 2 of the study, a similar error was also shown for LD- and MD samples.
During week 1 of sample analysis, only 2 females per group which are already treated with the test item (based on the measurements received lower doses as intended). After getting concern from the Sponsor, the study was kept on hold by stopping further mating of femlaes. However, two females (LD & MD groups) were dosed already, because those were pregnant. In week 2 sample analysis, first sample results was below the acceptane criteria, however repeat analysis showed measurements were within the acceptance criterion of 15%.
Apparently, the results which were outside of the acceptance criterion of 15% were based on the usage of plastic vials for the preparation of test item formulations which resulted in hydrolysis of the test item. After this issue was found, it was solved immediately (glass vials were used for preparation of test item formulations) for the remaining study period. Following results from analytical measurements were within the acceptance criterion of 15%.

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 104 sperm positive females, the remaining females and males were discarded without any observations.
- Further matings after two unsuccessful attempts: no
- 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.:

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

Remarks:

low dose (LD)

Doses / concentrations 2

Dose / conc.:

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

Remarks:

medium dose (MD)

Doses / concentrations 3

Dose / conc.:

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

Remarks:

high dose (HD)

No. of animals per sex per dose:

23 females in control group
24 females in low-, mid- and high-dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on the results from a dose range finding study, in which pregnant rats were treated with the test item at doses of 100, 300, 600 and 1000 mg/kg bw/day from GD 5 to 19. No mortality was observed in this dose range finding study up to 1000 mg/kg bw (the highest dose tested). Test item-related maternal toxicological effects in terms of body weight development, food consumption, mean terminal body (carcass) weight, net weight change and uterine weight were observed at 1000 mg/kg bw/day (HD group) when compared to the control group. Pre- and post-implantation loss was higher in the HD group than in controls. Consequently litter size was slightly smaller at the HD level than in controls. In the absence of test item-related external abnormalities, a test item related and statistically significant reduction in mean foetal weight was observed in both male and females foetuses at 300, 600 and 1000 mg/kg bw/day when compared to control. No external foetal abnormalities were noted, which were considered test item related. No maternal effects of dimethoxy(dimethyl)silane on were found at dose levels of 600 mg/kg bw/day and no effects of dimethoxy(dimethyl)silane on foetuses at 100 mg/kg bw/day were observed, respectively.
Based on this DRF study, high dose level of 1000 mg/kg bw/day was considered suitable for the main oral (gavage) prenatal developmental study. Furthermore, dose levels of 100 and 300 mg/kg bw/day were considered suitable as low and intermediate dose levels, respectively.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made at least once a day, preferably at the same time each day, except for female numbers 26 (LD), 51 (MD), and 76 (HD) where clinical observations were not made on GD 4, female numbers 23 (Control), 27 (LD), 29 (LD), 52 (MD) and 77 (HD) where clinical observation were not made on GD 20 and female no. 10 (Control) where clinical observation was not made from GD 1 to GD 4. 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.
- Clinical observations included: 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 or bizarre behaviour were recorded.

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 weighed in this study except once before initiation of pairing.

FOOD CONSUMPTION: Yes
- Food consumption of sperm positive females was measured on GD 5, 8, 11, 14, 17 and 20. Food consumption was not measured for males during the entire study or for both male and females during the mating period.


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: Uterus with the cervix and thyroid/parathyroid

OTHER: Thyroid hormone levels from samples from all dams were assessed at the end of the treatment as part of the sacrifice of the animals. At termination, blood samples were collected from the defined site in serum separator tubes and were stored under room temperature to collect serum. Stored serum samples (at ≤-20°C) were further processed and analysed 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:

Blood from the abdominal aorta of the animals was collected in serum separator tubes.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: Craniofacial examination of the heads of the fetuses used for the soft tissue examination of the first 20 litters per group, if possible.

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.

Historical control data:

Historical control data was provided for the following endpoints: uterine data, litter weight data, anogenital distance, maternal thyroid-related hormones, maternal thyroid/ parathyroid glands weight, fetal external examination, fetal visceral examination, fetal craniofacial examination, and fetal skeletal examination.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Female no. 91 of the HD group, showed clinical signs of piloerection, pale skin, and scratch/cut on the day of moribund sacrifice (GD 15).
Few specific clinical signs were observed in the MD and HD group. Hunched posture was noted in 1/24 females of the MD group, 2/24 females of the HD group and slow movements and ataxia was observed in 6/24 females of the HD group. These clinical signs were observed only on single day, however they are considered to be clinical signs of systemic toxicity.

Piloerection was observed in 2/24 females of the MD group and 5/24 females of the HD group on single days mostly at the end of the gestation phase. The clinical sign of piloerection is considered as general sign of discomfort or slightly reduced health condition but not systemic toxicity.

Moving the bedding was noted in 1/24 females of the LD group, 1/24 females of the MD group and in 4/24 females of the HD group on few days of treatment. Increased salivation was observed in 6/24 females of the HD group. Both signs were transiently seen after dose administration and were considered as a clinical sign elicited by local effects of the test item formulation and/or attributed to discomfort of the animals due to oral administration, but not systemic toxicity.

Local hairless areas on various body parts were noted in 1/23 females of the control group, 1/24 females of the LD group and 3/24 females of the HD group and the finding of a scratch/cut was observed in 1/24 females of the HD group. Both findings were considered incidental in nature.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Female no. 91 of the HD group was euthanized on GD 15 due to animal welfare reason. This animal showed piloerection, pale skin and scratch/cut on the day of sacrifice. Though, there were no severe maternal toxicity in other animals of HD group, this isolated mortality was most likely test item related effects.
All remaining females survived the scheduled study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weight increased in all groups before initiation of treatment and further increased with the progress of the study. However, mean body weight increase was dose-dependently lower in animals of test item-treated groups compared to the control group and was considered related to treatment with the test item.

After initiation of treatment, mean body weight gain was moderately lower during GDs 5 to 8 in the MD and HD groups when compared to controls (42% below control in MD and HD group). From GD 11, mean body weight gain remained moderately to slightly lower in all dose groups when compared to the control group with statistical significance noted in the HD group during GDs 14 to 17 (31% below control). When related to the entire study period (GDs 0 to 20), body weight gain was dose-dependently lower in the dose groups (10% below control in the LD group, 11% below control in the MD group) compared to the control group and resulted in a statistically significantly lower body weight gain in the HD group (19% below control).

Differences in body weight gain compared to controls resulted in slightly reduced mean body weight of the HD group from GD 8 onwards with the greatest effect on GD 20 (6% below controls) without achieving statistical significance. This slight effect on body weight development in HD group was considered as test item related.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption was lower in females of the dose groups compared to the control group on GD0-5 (14% below control in the LD group, 11% below control in the MD group and 18% below control in the HD group) and this may be considered to be cause of reduced body weight gain at later days of treatment period.

In correlation to lower body weight gain after initiation of treatment, mean food consumption tended to be dose-dependently lower in the dose groups compared to the control group from GD 5 to 8 (LD: 9%, MD: 10%, HD: 20% below control; only HD was statistically significant) and GD 8 to 11 (LD: 2%, MD: 9%, HD:11% below control) resulting in moderately and statistically significantly lower food consumption in the HD group compared to the control group from GD 5 to 8 and GD 11 to 14 (17% below control).

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid/parathyroid weight showed no statistically significant or biologically relevant effects when comparing dosing groups to the control group. Weights were comparable between the groups.

Uterine weight was slightly lower in all dose groups without dose dependency.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Female no. 91 from the HD group which was euthanized due to animal welfare reasons was noted with enlarged heart, pale lung, stomach, duodenum, jejunum, ileum, cecum, colon, liver and kidneys, a white spotted and enlarged spleen. Moreover, the animal showed a mass on the hind limb.
Female no. 59 from the MD group was observed with pale liver and kidneys and a placenta mass at necropsy. However, as no similar findings were observed in any of the other females of dose groups these findings were not considered test item related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The thyroid gland from 9 control, 13 low dose (LD), 17 medium dose (MD) and 18 high dose (HD) group animals showed minimal to slight follicular cell hypertrophy, while no changes were present in the parathyroid gland in any of the investigated animals at any dose level.
The incidence of the above mentioned follicular cell hypertrophy increased in a dose dependent manner reaching its highest level in the HD group. Further, the severity of the follicular cell hypertrophy was similar in control, LD and MD groups and slightly increased in the HD group (1.1 in HD group vs. 1.0 in all other groups including the control group). Therefore the above mentioned thyroid gland change was considered to be most likely test item related.
The thyroid gland induction occurs particularly with rodents, firstly because UDP-glucuronosyl transferase can easily be induced in rodents after exposure to various xenobiotics and, secondly, because thyroxine metabolism takes place very rapidly in rats in the absence of thyroxine-binding globulin (TBG). Therefore, the fast hepatobiliary clearance of thyroid hormones will lead to higher levels of circulating thyroid stimulating hormone (TSH) and to subsequent thyroid gland changes.
Based on the above mentioned particularities the observed thyroid follicular cell hypertrophy was considered to be most likely non-adverse.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Slightly and statistically significantly higher mean T3 levels were observed in the HD group compared to controls (3547 pg/mL in the HD compared to 2848 pg/mL in the controls). The mean TSH level was moderately higher in the HD group compared to the control group without achieving statistical significance (1679 pg/mL in the HD compared to 1073 pg/mL in the controls). The mean T4 levels showed no relevant difference between dose groups and the control group. As however, there was no dose dependency in mean T3, T4 or TSH hormones and all values were with in historical control range the effects of the test item on all the hormone levels were considered non adverse.

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

There were no biologically relevant or statistically significant effects on prenatal data.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Prenatal parameters like pre- and post-implantation loss remained unaffected in the dose groups when compared to the control group.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no biologically relevant or statistically significant effects on prenatal data.

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

Early resorptions remained unaffected in the dose groups when compared to the control group.
The incidence of late resorptions was statistically significantly higher in the HD group (% per animal, 6.43%) compared to the control group (% per animal, 0.48%) and the observed value is within historical control data. The mean incidence of late resorptions in the HD group was 0.61 compared to mean incidence of 0.05 in the control group. There were no late resorptions in the LD and MD groups.

Dead fetuses:

no effects observed

Description (incidence and severity):

No dead foetuses were observed in any of the test item-treated groups or the control group.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Successful mating resulted in 22/24 pregnancies in the LD group, 20/24 pregnancies in the MD group and 19/24 pregnancies in the HD group when compared to 19/23 pregnancies in the control group.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Maternal weight and adjusted maternal weight (carcass weight) on GD 20 (day of caesarean section) showed a slight dose-dependent trend towards lower mean weight in dose groups compared to the control group (maternal weight: MD: 3%, HD: 6% below control; adjusted maternal weight: LD: 1%, MD: 2%, HD: 5% below control). Uterine weight was slightly lower in all dose groups compared to the control group without following dose dependency (LD: 13%, MD: 8%, HD: 14% below control). The net weight change from GD 0 was dose-dependently reduced by 10%, 15% and 27% in the LD, MD and HD groups, respectively; the reduction was statistically significant at the HD group. The reduction in net weight change from GD 0 is considered to be test item related in all the dose groups.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

mortality

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Independent of the gender on per fetal basis, the mean foetus weight was found to be lower in the HD group which was considered test item-related. The mean male foetus weight was statistically significantly lower in the LD, MD and HD group compared to the control group {LD-3.56 g, MD-3.46 g, HD-3.14 g (4.28%, 7.08%, 15.6% respectively below control compared to 3.72 g in the control group}. Also the mean female foetus weight was statistically significantly lower in MD and HD groups compared to the corresponding controls (MD- 3.38 g and HD- 3.22 g (4.50% and 9% respectively, below control). This is considered to be test-item related due to the maternal toxicity which was found in the HD group in terms of altered body weight and food consumption of dams.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Live foetuses remained unaffected in the dose groups when compared to the control group.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio remained unaffected in the dose groups when compared to the control group.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

Independent of the gender on per litter basis, the mean foetus weight was found to be lower in the HD group (without statistical significance) which was considered test item-related; however there was a slight trend in lower mean foetal weight observed in MD group when compared to control. LD group was found to be comparable to control.

Anogenital distance of all rodent fetuses:

effects observed, treatment-related

Description (incidence and severity):

Male foetuses of the HD group showed statistically significantly shorter absolute AGD compared to male control foetuses (2.41 mm compared to 2.57 mm). However, as relative AGD shows no relevant difference it is assumed that shorter absolute AGD is based on the lower body weight of male foetuses. No statistically significant or toxicologically relevant effect on AGD was observed in female foetuses of any of the dose groups. Values were comparable between all groups.
Testicular (abdominal) descent was observed to be complete in all examined male foetuses with the exception of one male foetus from the HD group; this isolated finding was considered incidental. Thus, no effect of the test item on testicular descent was evident in test item-treated male foetuses.

Changes in postnatal survival:

not examined

External malformations:

effects observed, treatment-related

Description (incidence and severity):

At external examination, four foetuses from the HD group were observed with a discolored skin on single body parts resulting in statistically significantly higher foetal incidence of discolored skin in the HD compared to the control group (2% in the HD group compared to 0% in the control group). Due to the slightness, however, this finding was not considered adverse.

The finding of umbilicial hernia was observed in one foetus of the MD group and two foetuses of the HD group and hyperflexion of hindpaws was noted in one foetus of the LD group and two foetuses of the HD group. However, due to the low number of incidences these findings were not considered test item-related. Moreover, hyperflexion was not confirmed by malpositioned hindpaws during skeletal evaluation.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Skeletal examination and examination of cartilage of the Alizarin red and Alcian blue stained foetuses revealed a range of findings in all groups including control.
As usual for foetuses at this stage of gestation (day 20) incomplete ossification was seen in several bones of several litters in all groups including control. Mostly bones of the skull, sternum, paws and vertebra were affected by variations in the status of expected ossification in terms of incomplete ossification, misaligned ossification, irregular ossification, unossification or increased ossification throughout all groups. However, in this study a trend towards a test item-related incomplete ossification of several bones was observed which resulted in statistically significantly higher foetal incidences of incomplete ossification of numerous bones in the HD group.This is considered to be a secondary effect of lower foetal weight observed in the foetuses and maternal toxicity.
Bones showing statistically significantly higher foetal incidence of incomplete ossification in the HD compared to the control group were nasal bones (28% compared to 1%), frontal bones bilateral (45% compared to 6%) and right side (7% compared to 1%), parietal bones bilateral (64% compared to 17%), interparietal bone (88% compared to 55%), basioccipital bone (32% compared to 5%), exoccipital bone (10% compared to 0%), hyoid body (30% compared to 0%), basisphenoid bone (54% compared to 2%), alisphenoid bone (25% compared to 2%), zygomatic arch, bilateral (35% compared to 2%), squamosal (41% compared to 5%), clavicula (10% compared to 0%), ribs (30% compared to 0%), pelvic girdle ilium (29% compared to 1%) and pelvic girdle ischium (18% compared to 0%), cervical arches (58% compared to 2%), thoracic centra (24% compared to 0%), thoracic arches (24% compared to 0%), sacral centra (10% compared to 1%) and sacral arches (48% compared to 0%), caudal centra (7% compared to 0%) and caudal arches (34% compared to 0%), lumbar arches (25% compared to 0%), forelimb metacarpals (20% compared to 0%) and hindlimb metatarsals (37% compared to 1%), forelimb humeri (27% in the MD group and 69% in the HD group compared to 5% in the control group), ulnae (39% compared to 1%) and radii (29% compared to 2%), hindlimb femora (72% compared to 36%), tibiae (22% compared to 2%, without statistical significance) and fibulae (39% compared to 1%).Some other bones including the mandibles, zygomatic arch (left side) and pelvic girdle pubis showed a tendency towards lower ossification in dose groups, especially the HD group without achieving statistical significance.
For some bones a trend towards higher incidence of unossification was observed in dose groups compared to the control group. Foetal incidence for missing ossification of bones was statistically significantly higher in the dose groups (mostly HD group) compared to the control group for forelimb metacarpals (74% compared to 11%), hindlimb metatarsals (12% compared to 0%), cervical centra (98% in the MD group and 99% in the HD group compared to 74% in the control group), thoracic centra (11% compared to 0%), caudal centra (14% compared to 0) and caudal arches (36% compared to 0%).
Besides missing or incomplete ossification other statistically significant findings related to the ossification stage were observed especially for vertebra. Statistically significantly higher foetal incidence for dumbbell ossification (16% in the HD group compared to 0% in the control group), bipartite ossification (34% in the HD group compared to 0% in the control group), irregular ossification (62% in the MD group and 68% in the HD group compared to 20% in the control group), hemicentric ossification (10% in the HD group compared to 0% in the control group) and asymmetric ossification (16% in the HD group compared to 0% in the control group) was observed for thoracic centra in the MD and/or HD group which were considered test item-related. Irregular and hemicentric ossification of sacral centra were observed with a statistically significantly higher foetal incidence in the HD group compared to the control group (7% compared to 0% for both findings). For lumbar centra, statistically significantly higher foetal incidences in the HD group were observed for dumbbell ossification (10% compared to 0%), irregular ossification (28% compared to 0%), bipartite ossification (10% compared to 0%) and hemicentric ossification (6% compared to 0%).
The finding of split thoracic centra was observed with a statistically significantly higher foetal incidence in the HD group compared to the control group (5% compared to 0%). No split centra were observed in the LD or MD group.
Sternebra-associated findings were incomplete ossification, missing ossification, misaligned ossification and wide or split sternebra. The finding of incomplete ossification showed a slight trend towards higher incidences in dose groups with statistical significance in the HD group compared to the control group for 1st sternebra (7% compared to 0%), 2nd sternebra (51% compared to 5%), 3rd sternebra (10% compared to 0%) and 4th sternebra (11% compared to 1%). A higher foetal incidence of unossified sternebra was observed in the HD group with statistical significance for 2nd sternebra (12% compared to 0%), and 6th sternebra (63% compared to 3%). Missing ossification of 1st, 3rd, 4th and 5th sternebra showed no statistically significant or toxicologically relevant differences in dose groups compared to the control group. The incidence of misaligned, wide or split sternebra in dose groups was generally observed with low incidences and in rates comparable to the control group without statistical significance and thus was not considered toxicologically relevant.
Increasing incidence of misshapen/bent/thick bones was observed in dose groups resulting in statistical significant differences of some findings which were considered test item-related. The foetal incidence of misshapen humeri showed a dose-dependent trend resulting in a statistically significantly higher foetal incidence in the HD group (foetal incidence C: 0%, LD: 3%, MD: 7%, HD: 32%). Thick and bent humeri were observed without statistical significance or dose-dependency in two foetuses of the LD and one foetus of the HD group (thick) and three foetuses of the HD group (bent). Misshapen ulnae showed a foetal incidence of 1% in the MD group and statistically significantly higher foetal incidence of 12% in the HD group compared to 0% in the control group. Bent ulnae were observed without dose-dependency or statistical significance with a foetal incidence of 3% in the control group, 2% in the LD group, 0% in the MD group and 9% in the HD group. The foetal incidence of misshapen radii was statistically significantly higher in the HD group compared to the control group (8% compared to 0%) without occurrence of misshapen radii in the LD and MD group and foetal incidence of bent radii showed a dose-dependently increasing trend with statistical significance in the HD group (C: 1%, LD 1%, MD: 3%, HD: 23%). Foetal incidence of bent pelvic girdle ilium was statistically significantly higher in the HD group (7%) compared to the control group (0%) without occurrence of bent ischia in the LD and MD group. The foetal incidence of misshapen femora showed a slight dose-dependent increase (0% in control, 1% in LD, 2% in MD) resulting in statistically significantly higher foetal incidence in the HD group (23%). The incidence of bent femora was 1% in the LD group and 4% in the HD group. Misshapen tibiae showed a higher foetal incidence of 2% in the MD group and statistically significantly higher incidence of 21% in the HD group compared to the controls whereas no incidences occurred in the control and LD group. Foetal incidence of bent tibia was marginally higher in the MD group compared to the control group (1% compared to 0%) and statistically significantly higher in the HD group (11%). Foetal incidence of bent fibulae was statistically significantly higher in the HD group compared to the control group (12% compared to 0%) and slightly higher in the MD group (2%) without occurrence of this finding in the control and LD group. The incidence of misshapen fibulae was slightly higher in the HD group compared to the control group (6% above control) without any incidences of misshapen fibulae in the control, LD and MD group.
Bent scapulae were observed with a slight dose-dependent trend resulting in a moderately but statistically significantly higher foetal incidence in the HD group compared to the control group (51% in the HD group compared to 3% in the control group). Foetal incidence of misshapen scapulae was observed to be statistically significantly higher in the HD group (10%) compared to the control group (0%). The finding of bent scapular spine showed a slightly higher foetal incidence in the MD group (12%) and statistically significantly higher incidence in the HD group compared to the controls (32% compared to 2%). The finding of bent scapula appears to be transient and completely repaired post-natally [Kimmel et al.; 2014; Hofmann et al. 2016] and thus may be classified as variation.
Supernumerary (14th) thoracolumbal ribs (right side, left side and rudimentary or full) were noted in several foetuses throughout all groups including the control group control. The incidence of unilateral supernumerary ribs and bilateral rudimentary ribs showed no dose-dependent trend or statistical significance in dose groups when compared to the control group. The incidence of bilateral supernumerary full ribs, however, was observed with a dose-dependent trend in foetal and litter incidence resulting in statistically significantly higher foetal incidence of supernumerary full ribs in the HD group compared to the control group (49% in the HD group compared to 4% in the control group) which was considered test item-related.
Cervical ribs (right side, left side, bilateral and rudimentary or full) were noted in a few foetuses throughout all dose groups with a slight dose-dependent trend towards higher incidence of cervical ribs with increasing dose of test item. Rudimentary ribs (bilateral) showed a foetal incidence of 0% in the control group, 1% in the LD group, 4% in the MD group and 9% in the HD group. Rudimentary ribs (right side) were observed with a foetal incidence of 0% in the control and LD group, 2% in the MD group and statistically significantly higher foetal incidence of 13% in the HD group. Rudimentary ribs (left side) showed foetal incidences of 0% in the control group, 2% in the LD group, 8% in the MD group and statistically significantly higher foetal incidence of 10% in the HD group. Full cervical ribs (bilateral) were observed with a foetal incidence of 3% in HD group compared to 0% in the control, LD and MD group. Cervical ribs (right side) showed a foetal incidence of 0% in the control and LD group, 2% in the MD group and 5% in the HD group and left side cervical ribs were observed with a foetal incidence of 0% in control and LD group, 2% in the MD group and statistically significantly higher foetal incidence of 15% in the HD group. In general, cervical ribs are often transient and produce no adverse effects on animal survival or health of this strain and age. Short (rudimentary) ribs are considered as variations (Monograph No. 31, 2002). Wavy ribs were observed at an incidence of 29% in the control group, 43% in the LD group and statistically significantly higher incidence of 72% in the MD group and with 90% also in the HD group. In general, wavy ribs are typically classified as transient and reversible post-natally and thus may be considered as variations but not malformations [Kimmel et al., 2014, DeSesso and Scialli, 2018].
Foetal incidence of pelvic girdle caudal shift (bilateral) increased dose-dependently compared to the control group (10% in the control group, 12% in the LD group, 18% in the MD group, 66% in the HD group). Pelvic girdle caudal shift (left side / right side) was noted in few foetuses throughout all groups including control without statistically significant difference of dose groups to controls and without a dose-dependent trend. Although the incidence of caudal shift of the pelvic girdle is increasing with the test item dose it is not considered adverse as changes of the position of the pelvic girdle relative to the number of pre-pelvic vertebrae can occasionally be seen in animals of this strain.
Other observed ossification-related findings were observed at low incidences and showed no statistically significant differences or dose-dependency and thus were not considered toxicologically relevant.
The HD group was observed with a statistically significantly higher foetal incidence of supernumerary cervical cartilaginous ribs (43% in the HD group compared to 0% in the control group) and a statistically significantly higher foetal incidence of cervical cartilaginous ribs fused to the first costal cartilage (22% in the HD group compared to 0% in the control group. Both findings were considered test item-related.
Foetal incidence of supernumerary costal cartilage was dose-dependently higher in dose groups (6% in control, 19% in LD and 27% in MD) resulting in statistically significantly higher foetal incidence in the HD group compared to the control group (53%). Remaining findings related to the costal cartilage (long, interrupted, fused, hooked at tip, nodulated at tip, branched at tip, flattened) were generally at frequencies comparable between the dose groups and the control group and were not considered toxicologically relevant.
The finding of wide intersternebral cartilage showed a statistically significantly higher foetal incidence in the HD group compared to the control group (35% compared to 7%) whereas frequencies of wide intersternebral cartilage in the LD and MD group was comparable to the control group. Foetal incidence of split intersternebral cartilage was observed with statistically significantly higher incidence in the HD group compared to the control group (14% compared to 2%). The incidence of fused cervical cartilaginous arches showed a trend towards higherincidence in dose groups compared to the control group resulting in statistically significantly higher incidence in the HD group (6% in control group, 7% in LD group, 22% in MD group and 46% in HD group). The cervical cartilaginous ventral plate was observed with increased incidences of cranial shift (2% in the control group, 3% in the LD group, 4% in the MD group, 11% in the HD group), cranial expansion (4% in the control group, 3% in the LD group, 7% in the MD group and 14% in the HD group) or long appearance (8% in the control group, 5% in the LD group, 17% in the MD group, 20% in the HD group) in the MD and HD group without achieving statistical significance in any of the groups.
There were also slightly higher foetal incidences of skeletal findings in LD and MD groups, though they did not show statistically significant changes and/or many of these findings were not within historical control range for this strain, hence it considered to be test item related findings.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

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 compared to controls.
The finding of supernumerary liver lobe was observed in statistically significantly more litters in the HD group (10 litters affected) compared to the control group (2 litters affected) and with a higher foetal incidence in the HD group (15% foetal incidence) compared to the control group (2% foetal incidence). It cannot be excluded that treatment with the test item caused the higher incidence of supernumerary liver lobes in the HD group, however, this change is considered a variation rather than malformation and thus is not assumed adverse.
A comparable incidence of malpositioned umbilical artery was observed between HD and control groups (litter incidences: C: 63%, LD: 45%, MD: 47%, HD: 65%), lower incidences of bilateral azygos vein in all dose groups when compared to control group (litter incidences: C: 42%, LD: 20%, MD: 32%, HD: 12%), lower incidence of malpositioned testis in LD and HD groups when compared to control group (litter incidences: C: 42%, LD: 10%, MD: 42%, HD: 35%), higher incidence of dilated renal pelvis in all dose groups when compared to control group (litter incidences: C: 26%, LD: 45%, MD: 32%, HD: 47%) and higher incidence of long thymus in HD group when compared to control group (litter incidences: C: 32%, LD: 5%, MD: 21%, HD: 53%). However, none of these findings showed a dose-dependent trend towards a higher incidence in dose groups compared to the control group.
The finding of an internal abdominal haemorrhage was observed with a litter incidence of 65% in the LD group, 63% in the MD group and 59% in the HD group compared to 47% in the control group and subcutaneous haemorrhage at the neck was noted with a litter incidence of 100% in the LD group, 95% in the MD group, 59% in the HD group compared to 90% in the control group. The incidence of both findings followed no dose dependency and was considered to be the result of the handling of the foetuses during caesarean sections and evaluations and thus not toxicologically relevant.
Isolated findings including malpositioned azygos vein in two foetuses of the LD and one foetus of the HD group, malpositioned origin of the subclavian artery in one foetus of the control group and one foetus of the MD group, supernumerary artery in one foetus of the MD group, dilated ureter in one foetus of the MD group, large liver in one foetus of the HD group, small testis in one foetus of the HD group, malpositioned kidneys in one foetus of the MD group, and small spleen in one foetus of the LD and two foetuses of the HD group were not considered toxicologically relevant due to their low incidence and the lack of dose dependency.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

At craniofacial examination no abnormalities of toxicological relevance were observed in any of the test item-treated groups. Statistical analysis of foetal and litter incidences showed no significant differences compared to the control group.
Single findings of subcutaneous edema in two foetuses of the HD group, subcutaneous haematoma in one foetus of the MD group, subdural haematoma in one foetus of the MD group and discolored cerebrum in one foetus of the control group were not considered toxicologically relevant due to the lack of dose dependency and the generally low number of incidences.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Fetal abnormalities

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: decreased fetal weight

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

The study was conducted according to OECD 414 test guideline, and in compliance with GLP for the registered substance. On the basis of decreased mean body weight gain, decreased mean carcass weight (net weight change from gestation day 0) at all the dose levels and mortality at HD group, the NOAEL for maternal toxicity is considered to be less than 100 mg/kg bw/day. Based on decreased fetal weight, the NOAEL for foetal toxicity is considered to be 100 mg/kg bw/day.