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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020/11/22 - 2021/01/05
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)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Triethoxy(3-thiocyanatopropyl)silane
EC Number:
252-161-3
EC Name:
Triethoxy(3-thiocyanatopropyl)silane
Cas Number:
34708-08-2
Molecular formula:
C10H21NO3SSi
IUPAC Name:
3-(triethoxysilyl)propyl thiocyanate
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar Han
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Deutschland, Sulzfeld
- Age at study initiation: 11-15 weeks
- Weight at study initiation: 176-273g
- Fasting period before study: No
- Housing: Individually in Macrolon plastic cages (MIII type)
- Diet: SM R/M-Z from SSNIFF, ad libitum
- Water: Municipal tap water, ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21
- Humidity (%): 51-55
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2020/11/17 To: 2020/12/10

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Remarks:
dried and de-acidified arachis oil, specific gravity 0.885
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 6 hours after adding the vehicle to the test item.

Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.

Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle: Trial preparations (not part of this study) were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure
- Concentration in vehicle: 0, 31.25, 62.5, and 125 mg/mL
- Amount of vehicle: Dosing solution volume, 4 mL/kg bw
- Lot/batch no.: Not specified
- Purity: Not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis during Week 1 of treatment, with analysis of concentration (all groups) and homogeneity (125 and 500 mg/kg bw/day groups). Analyses were performed using a validated analytical procedure.
Details on mating procedure:
The females arrived on Day 0 or Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating).
Duration of treatment / exposure:
Day 6 to 20 post-coitum, inclusive
Frequency of treatment:
Once daily
Duration of test:
Day 0 or 1 post coitum through sacrifice on Day 21 post-coitium
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
22F
Control animals:
yes
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Two range-finding studies, one with time-mated females and one with nulliparous and non-pregnant females
- Rationale for animal assignment: Random

Examinations

Maternal examinations:
MORTALITY AND MORBIDITY: Yes
Animals were observed for mortality and morbidity twice daily, in the morning and at the end of the working day.

CAGE SIDE OBSERVATIONS: Yes
Once daily, beginning on Day 6 post-coitum onwards up to the day prior to necropsy.

DETAILED CLINICAL OBSERVATIONS: Yes
Weekly, beginning during the Pre-treatment Period, and on the day of necropsy.

BODY WEIGHT: Yes
On Days 2, 6, 9, 12, 15, 18 and 21 post-coitum. In order to monitor the health status, Female No. 7 (control) was also weighed on Day 13 post-coitum.

FOOD CONSUMPTION: Yes
Quantitatively measured over Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum.

WATER CONSUMPTION: Yes
Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

CLINICAL CHEMISTRY: Yes
Blood samples were collected on the day of scheduled necropsy (animals were not fasted) and analysed for thyroid hormone: triiodothyronine (T3), thyroxine (T4), and thyroid-Stimulating Hormone (TSH).

SACRIFICE:
Post-coitum Day 21, by carbon dioxide inhalation.

Terminal procedures are summarized in Table 1 below.

ORGAN WEIGHT: Yes
The thyroid and uterus were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead. Paired organs were weighed together. Organ to body weight ratios (using the body weight on Day 21 post-coitum) were calculated.

GROSS NECROPSY: Yes
All animals (including Female Nos. 76 and 83 (500 mg/kg/day) that were found dead) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin.

HISTOPATHOLOGY: Yes
Thyroid gland was examined histopathologically. No macroscopic abnormalities were identified, thus no further tissues were subjected to histopatholgy.
Ovaries and uterine content:
The ovaries and uterine content were examined after termination, 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
- Number and distribution of live and dead fetuses: Yes
Fetal examinations:
SACRIFICE:
By administration of sodium pentobarbital into the oral cavity using a small metal feeding tube. Due to a malformation for Fetus No. 50-L1 (250 mg/kg/day), this fetus was euthanized by intrascapular injection of sodium pentobarbital.

FETAL EXAMINATION: Yes
Litters of females surviving to scheduled necropsy were subjected to detailed external, visceral and skeletal examinations. For recognizable dead embryos in development for the two females found dead on Day 9 post-coitum (500 mg/kg bw/day), no gross external examination was performed.

EXTERNAL EXAMINATION: Yes
Each viable fetus was sexed, examined in detail to detect macroscopic visible abnormalities and their weight (not for fetuses of animals found dead) was determined. The anogenital distance (AGD) was measured for all viable fetuses. The AGD was normalized to the cube root of the fetal body weight.

VISCERAL EXAMINATION: Yes
The sex of all fetuses was confirmed by internal examination and approximately one-half of the fetuses (live and dead) in each litter (all groups) were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar.

The heads were removed from this one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination using the Wilson sectioning technique.

Tissues with variations or malformations were stored in 10% formalin.

SKELETAL EXAMINATION: Yes
Skeletal examination was done for one-half of the fetuses (i.e. the fetuses with heads). A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing (listed in raw data).
Statistics:
Means, standard deviations (or % coefficient of variation or standard error, when deemed appropriate), ratio, percentages, numbers, and/or incidences were reported as appropriate by dataset. All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. Pairwise comparisons were made for each test substance dose group (125, 250, and 500 mg/kg bw/day) versus controls.

A combination of parametric and non-parametric methods were used, and collectively included: Levene’s test, one-way ANOVA F-test, Kruskal-Wallis test, Dunnett’s or Dunn’s test, analysis of covariance (ANCOVA), Fisher’s exact test.
Indices:
MATERNAL:
- Pregnancy Rate (%): No. of pregnant females x 100 / No. of mated females

FETAL:
- Male Fetuses (%): No. male fetuses x 100 / No. of fetuses
- Female Fetuses (%): No. female fetuses x 100 / No. of fetuses
- Pre-Implantation Loss (%): (No. of corpora lutea – No. of implantations) x 100 / No. of corpora lutea
- Post-Implantation Loss (%): (No. of implants – No. of live fetuses) x 100 / No. of implantations
- Litter % of Fetuses with Abnormalities: No. of fetuses in litter with a given finding x 100 / No. of fetuses in litter examined
Historical control data:
Yes, for these endpoints:
- Clinical chemistry, Wistar Han female rats (period 2020-2021)
- Fetal body weight, Wistar Han F1 rats (period 2016-2020)
- Skeletal variations, Wistar Han Rats (period 2015-2020), including supplemental data for the same rat strain from another Charles River facility

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
These test substance related clinical signs were observed during varied periods of the study:
- decreased activity (250 and 500 mg/kg bw/day)
- closure of left and rights eyes (500 mg/kg bw/day).

The relationship to the test substance was not specified for erect fur (250 and 500 mg/kg bw/day) and hunched posture (500 mg/kg bw/day).

Other clinical signs were identified as no toxicological relevance.
Mortality:
mortality observed, treatment-related
Description (incidence):
Test item-related mortality was observed at 500 mg/kg bw/day consisting of two females that were found dead on Day 9 post-coitum. No signs of toxicity were noted for one female on previous days, while the other female exhibited salivation, erected fur and decreased activity on previous days. For both animals, no abnormalities during necropsy were observed that could explain their spontaneous deaths. However, both mortalities were considered adverse as these were observed only in the high dose group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain was affected by treatment with the test item at 500 mg/kg bw/day.

Mean body weight gain was decreased (52% of control) at 500 mg/kg bw/day between Days 6-9 post-coitum and remained minimally lower throughout the treatment period (not statistically significant from Day 9 onwards). Mean body weight at the end of the treatment period was slightly decreased (3%) at 500 mg/kg bw/day (not statistically significant).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was considered affected by treatment with the test item at 500 mg/kg bw/day.

Mean food consumption was lower compared to control group values (-14.80% and -9.52% of control) at 500 mg/kg bw/day between Days 6-9 and 9-12 post-coitum, respectively, with normal food consumption over the periods thereafter.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Description (incidence and severity):
Water consumption was monitored by visual inspection of water bottles/containers, results not reported.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Serum levels of total thyroxine (T4) were considered affected by treatment with the test item starting at 250 mg/kg bw/day.

Mean serum levels of total T4 were decreased (0.85x and 0.83x of control) at 250 and 500 mg/kg bw/day, respectively, but remained within the historical control range.

The findings for TSH at 125, 250, and 500 mg/kg bw/day were considered of no toxicological significance (based on no dose-response, high mean historical control), while T3 was unaffected by treatment up to 500 mg/kg bw/day.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test item-related alterations in thyroid weights up to 500 mg/kg bw/day.

Mean gravid uterus weight was considered unaffected by treatment with the test item up to 500 mg/kg bw/day.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related macroscopic findings up to 500 mg/kg bw/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Thyroid gland, follicular cell hypertrophy was present at increased incidence (6/22) in females treated at 500 mg/kg/day at minimal degree. The incidences at other doses were 1/22 at 0 mg/kg/day, 2/22 at 125 mg/kg/day, and 2/22 at 250 mg/kg/day.

See Table 2 below.
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
not examined
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The numbers of pre- and post implantation loss in the control and test groups up to 500 mg/kg bw/day were similar and in the range of normal biological variation.
Total litter losses by resorption:
not examined
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
Compared to controls, number of early (and total) resorptions were 50% lower, 35.7% lower, and 29.3% lower at 125, 250 and 500 mg/kg bw/day, respectively. A dose-relationship to test-item treatment is not evident.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
At scheduled necropsy, all females were found to be pregnant with viable fetuses. The numbers of pregnant females in the control and test groups up to 500 mg/kg bw/day were similar and in the range of normal biological variation. Two females (Nos. 76 and 83) at 500 mg/kg bw/day did not survive until scheduled necropsy.
Other effects:
no effects observed
Description (incidence and severity):
The numbers of corpora lutea and implantation sites in the control and test groups up to 500 mg/kg bw/day were similar and in the range of normal biological variation.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
mortality

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Fetal body weights were affected by treatment with the test item at 500 mg/kg bw/day.

Mean fetal body weights were decreased (-9.0%, -8.6% and -8.8% of control) at 500 mg/kg bw/day in males, females and combined sexes, respectively, which were below the historical control range.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
Compared to controls, number of live fetuses were 1.6% higher, -2.0% lower, and -1.3% lower at 125, 250 and 500 mg/kg bw/day, respectively. A clear dose-relationship to the test-item is not evident.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio was not affected by treatment with the test item up to 500 mg/kg bw/day.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Litter size was not affected by treatment with the test item up to 500 mg/kg bw/day.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No external malformations or variations were noted among fetuses that were considered to be related to treatment with the test item up to 500 mg/kg bw/day.

See Tables 3 and 4 below.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Skeletal malformations were noted among fetuses that were considered to be related to treatment with the test item starting at 125 mg/kg bw/day.

Skeletal malformations were noted in 1 (1), 3 (3), 2 (2) and 4 (3) fetuses (litters) of the control, 125, 250 and 500 mg/kg bw/day groups, respectively.

Among skeletal malformations, a higher mean incidence of fetuses for a supernumerary vertebra (1.1%, 1.8% and 0.83%) was observed at 125, 250 and 500 mg/kg bw/day, respectively, while this finding was not observed in the concurrent control or historical controls of the Test Facility. However, it has been recorded in the historical control data at the Charles River Lyon facility (which uses rats of the same strain) at a fetal incidence of 0.82%. At the incidence observed, a test item-relationship could not be excluded.

Three fetuses (67 R10, 67-R14 and 85 R9) at 500 mg/kg bw/day also had a rib anomaly which was considered a secondary effect since it occurred in conjunction with the vertebral anomalies.

Among skeletal variations, mean litter incidences of a pelvic girdle with misaligned ilium were higher in test item-treated groups compared to control. Mean litter incidences of fetuses with this finding were 1.9%, 5.8%, 7.5% and 10.7% in the control, 125, 250 and 500 mg/kg bw/day groups, respectively, which were not statistically significant and remained within the historical control range.

Given their low incidences and/or in the absence of a dose-related trend, all other skeletal malformation and variations were considered not related to treatment with the test item.

See Tables 3 and 4 below.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No visceral malformations or variations were noted among fetuses that were considered to be related to treatment with the test item up to 500 mg/kg bw/day.

See Tables 3 and 4 below.
Other effects:
no effects observed
Description (incidence and severity):
AGD (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment with the test item up to 500 mg/kg/day.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Remarks on result:
other: Based on decreased fetal body weight at 500 mg/kg bw/day
Remarks:
Effects econdary to maternal toxicity

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
500 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

Any other information on results incl. tables

Table 2. Summary test item-related thyroid gland findings a

Dose Level (mg/kg/day):

Females

0

125

250

500

 

 

 

 

 

THYROID GLANDS b

22

22

22

20

   Follicular cell hypertrophy

 

 

 

 

      Minimal

1

2

2

6

a Excluding animals found dead (2/22 at 500 mg/kg bw/day)

b Number of tissues examined from each group

Table 3. Number of fetuses (litters) submitted to the different examinations

Dose level (mg/kg/day):

0

125

250

500

External examination

252 (22)

256 (22)

247 (22)

224 (20)

Visceral examination

127 (22)

127 (22)

124 (22)

112 (20)

Skeletal examination

125 (22)

129 (22)

123 (22)

112 (20)

 

Table 4. Summary of malformations - individual descriptions

Dose Level (mg/kg bw/day)

 

Female No.

 

Fetus No.

 

Malformation(s)a

0

2

L2

Sternoschisis (S)

125

23

L4

Lumbar vertebra, 1 or more, Supernumerary (S)

26

L4

Thoracic arch, 1 or more, Fused (S)

Thoracic centrum, 1 or more, Absent (S)

42

R6

Thoracic arch, 1 or more, Absent (S)

Thoracic centrum, 1 or more, Absent (S)

250

45

R9

Lumbar vertebra, 1 or more, Supernumerary (S)

50

L1

Jaw, lower (Mandible), Absent (E)

Mouth, Absent (E)

General, Situs inversus (V)

Lung Lobe, Right cranial, Fused (V)

53

L5

Lumbar vertebra, 1 or more, Supernumerary (S)

500

67

R10

Rib, 1 or more, Fused (S)

Thoracic vertebra, 1 or more, Absent (S)

R14

Rib, 1 or more, Supernumerary (S)

Thoracic centrum, 1 or more, Absent (S)

84

R6

Lumbar vertebra, 1 or more, Supernumerary (S)

85

R9

Rib, 1 or more, Absent (S)

Thoracic arch, 1 or more, Absent (S)

Thoracic centrum, 1 or more, Absent (S)

a Including external (E), visceral (V) and skeletal (S) examinations.

Applicant's summary and conclusion

Conclusions:
In a study conducted according to OECD Test Guideline 414 and in compliance with GLP (reliability score 1), pregnant Wistar Han rats were administered triethoxy(3-thiocyanatopropyl)silane at 0, 125, 250, and 500 mg/kg bw/day via oral gavage (arachis oil vehicle) during days 6 to 20 post-coitum, inclusive. The maternal NOAEL was 250 mg/kg bw/day, based on mortality at 500 mg/kg bw/day. The fetal NOAEL was 250 mg/kg bw/day, based on decreased fetal body weight 500 mg/kg bw/day. However, this finding was likely to be secondary to maternal toxicity at the high dose, which was above the maximum tolerated dose. For fetal skeletal malformations, a test item-relationship could not be excluded for a supernumerary vertebra (observed at 125, 250 and 500 mg/kg bw/day). No fetal external or visceral malformations or variations were considered treatment-related up to 500 mg/kg bw/day.