Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422), rat:

NOAEL(reproductive toxicity) = 250 mg/kg bw/day

NOAEL(systemic toxicity) = 250 mg/kg bw/day

OECD 443: This information will be submitted later based on ECHA decision number CCH-D-2114428794-40-01/F.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl: CD® (SD) IGS BR VAF/Plus®
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC 27610
- Age at study initiation: 9 weeks at experimental start
- Weight at study initiation: 192 to 239 g at experimental start (Females). 283 to 341 g at experimental start (Males)
- Housing: Animals were individually housed in suspended wire-mesh cages elevated above faecal pans containing Bed-O’ Cobs® litter, during quarantine/ acclimation and during the in-life phase of the study. Animals were given Nylabones® and Cozee Pads for environmental enrichment while in standard housing. Environmental enrichment was removed from the toxicity group male and female animals the afternoon/evening prior to necropsy.
- Diet: Lab diet 5002, Certified Rodent Diet (PMI Nutrition International) was provided ad libitum during the quarantine/acclimation period and throughout the study. Food was removed the afternoon/evening prior to necropsy of toxicology animals
- Water: Municipal water, further purified by reverse osmosis was available ad libitum via automatic watering system.
- Acclimation period: 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 -21.7
- Humidity (%): 53-59
- Air changes (per hr): 14.3
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 20-10-2008 To: 30-07-2009.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Each dosing solution was prepared individually. Dosing solutions were prepared by adding the appropriate amount of the test article to a tare container and adding the appropriate amount of corn oil to yield the desired dose level. Dosing solutions were administered by oral gavage with a 100 mm, 15 gauge (1.8 mm), plastic feeding tube and syringe. The test article was administered at a dose volume of 4 ml/kg bw and was calculated from the most recent body weight.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Based upon the physical and chemical properties of the test article, dried and deacidified corn oil was considered to be the most appropriate vehicle for oral administration.
- Lot/batch no. (if required): 058K0070
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Study day 15 up to Study day 28
- Proof of pregnancy: Females rats were evaluated daily for evidence of copulation, by confirming the presence of either vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy.
- After successful mating each pregnant female was caged (how): Day 0 of gestation was defined as the day evidence of copulation was observed, at which time the female was individually caged.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosing solution analysis was performed by a GC/FID method to verify concentration, stability, and homogeneity of the test article in carrier. Concentration verification was conducted for the initial and third dose preparations.
Duration of treatment / exposure:
Male rats were administered the test substance for 29 consecutive days, while female rats were administered the test substance up to 51 days in total. Female rats were exposed for a two-week pre-mating phase, a 1-14 day mating phase, and through day post-partum, up to 51 days in total.
Frequency of treatment:
Daily
Details on study schedule:
Not reported
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:Clinical observations were performed daily immediately following exposure.

BODY WEIGHT: Yes
- Time schedule for examinations:Individual body weights were determined beginning with randomisation into test groups, on the first day of dosing, at least weekly thereafter, and on the day of euthanasia. During gestation, the reproductive group females were weighed (at a minimum) on gestation days 0, 7, 14 and 20, within 24 hours of parturition and on day 4 post-partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes


Oestrous cyclicity (parental animals):
Not reported
Sperm parameters (parental animals):
Parameters examined in [P] male parental generations:
[testis weight, epididymis weight, sperm morphology, ]
Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in [F1 ] offspring:
[number and sex of pups, stillbirths, live births, runts, and the presence any gross anomalies. Live pups were counted, sexed and the sex ratio calculated]

GROSS EXAMINATION OF DEAD PUPS:
[yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.]
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals [After a minimum of 28 days on study]
- Maternal animals: All surviving animals were euthanised by CO2 inhalation on post-partum day 4.

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGHTS
Tissues and organ taken for histopathological examination:
Adrenal, Brain (including cerebrum, cerebellum and pons), Cecum, Colon, Duodenum, Epididymis, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Lymph nodes, Ovaries, Prostate, Sciatic nerve, Seminal vesicles, Spinal cord, Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid, Trachea, Urinary bladder and Uterus (horns and cervix).
The following organs were weighed:
Adrenal, Brain (including cerebrum, cerebellum and pons), Epididymis, Heart, Kidneys, Liver, Seminal vesicles, Spleen, Testes, Thymus,and Uterus (horns and cervix).
Postmortem examinations (offspring):
Not applicable
Statistics:

For reproductive endpoints that involve occurrence up to birth (corpora lutea counts, total implants, post-implantation losses, day’s gestation) and for the total number of pups in the litter and total live pups in the litter, an ANOVA was done. If a statistically significant difference across treatment groups was seem pair-wise comparisons are made between the control group and the treated groups using Dunnett’s tests. For the remaining endpoints an ANOVA with the treatment and total litter size as independent variables was done. For all these analyses total litter size is defined as the total number of pups, both alive and dead. If the treatment was a significant variable in the analysis, then pair-wise comparisons were made between the control and each treated group using Dunnett’s test and adjusting for the litter sizes.
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not specified
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
effects observed, treatment-related
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
All but one animal survived to their scheduled necropsy. For the males at 1000 mg/kg bw/day, significant abnormal observations (p<0.01) were noted and included soiling of the chin and urogenital area. Abdominal, chin, muzzle and urogenital soiling were significant abnormal observations (p<0.01) in the reproductive group females at 1000 mg/kg bw/day.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There were no statistically significant differences between controls and treatment groups in the mean body weights on any of the test groups.
There were no differences in the average daily food consumption between controls and the males groups for any of the measured time periods. In the reproductive group females, at 1000 mg/kg bw/day, there was a significant decrease (35%) in food consumption from control s during the intervals post-partum days 0-4.


REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
In the testes there was moderate to marked seminiferous tubule degeneration observed in all 1000 mg/kg bw/day male rats that was characterised by degeneration of spermatocytes. A downstream effect was observed in the epididymides of the same rats. This is an adverse finding.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
In the reproductive group females, there was no statistically significant difference across treatment groups for corpora lutea and total implants. However, there was statistically significant difference at 1000 mg/kg bw/day for post-implantation losses, days of gestation, total pups and total live pups with the 1000 mg/kg bw/day groups having significantly more post-implantation losses, a longer gestation period, fewer pups in the litter and fewer live pups in the litter than were seen in the control.

ORGAN WEIGHTS (PARENTAL ANIMALS)
The increased liver weights in males and females at 1000 mg/kg bw/day correlated with the histopathologic finding of panlobular hypertrophy and centrilobular hypertrophy in females at 250 mg/kg bw/day. In males, adrenal cortical atrophy was accompanied by a decrease in absolute and relative adrenal weights at 1000 mg/kg bw/day, seminiferous tubule degeneration was accompanied by a decrease in absolute and relative testes weights at 1000 mg/kg bw/day, epididymal effects were accompanied by a decrease in absolute and relative decreases in epididymal weights, and kidney nephropathy findings were accompanied by an increase in relative kidney weights.


GROSS PATHOLOGY (PARENTAL ANIMALS)
The ovaries appeared enlarged in one female/group administered ≥50 mg/kg bw/day. The seminal vesicles appeared small in 4 males administered 1000 mg/kg bw/day, and in one animal each at the 50 and 250 mg/kg bw/day dosage levels.

Testes:
There was moderate to marked seminiferous tubule degeneration observed in all 1000 mg/kg bw/day male rats, significant at p<0.01. Minimal seminiferous tubule degeneration was recorded for one male rat dosed at 250 mg/kg bw/day (not statistically significant). The finding was not observed in control rats or those administered 50 mg/kg bw/day. The finding was characterised by degeneration of spermatocytes that appeared to be arrested and dying while in meiotic division (to become spermatids) or degenerating at earlier spermatocytes stages. Because of cell death at the meiotic spermatocytes stage, meiotic spindles were observed much more commonly in the high-dose males than in controls.

Epididymides:
The beginning of the downstream effect of the spermatocyte degeneration in the testes was observed in the epididymides of all 1000 mg/kg bw/day male rats. A mild increase in immature spermatids was observed throughout the epididymides in 10/10 animals, significant at p<0.01 however they were especially common in the head portion. A minimal number of immature spermatids were observed in one 250 mg/kg bw/day male rat. Mild to moderate hypospermia, statistically significant at p<0.01, in 9/10 animals was also observed at the highest dosage, but it should be noted that this was largely confined to the head of the epididymides. Sperm numbers appeared to be near normal in the remainder of the epididymides.



Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: At 1000 mg/kg bw/day- in males: hepatic protoporphyrin accumulation, adrenal cortical atrophy, kidney protein droplet nephropathy, testicular seminiferous tubule degeneration with epididymides involvement and in females rats, periportal vacuolation
Critical effects observed:
not specified
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
The number of day 4 viable pups and the ratio of the number of viable pups to the total litter size was significantly different, with the 1000 mg/kg bw/day group having fewer viable pups by day 4 and a smaller viable/total ratio than did the control group. The percentage of post-natal loss was significantly different, with the 1000 mg/kg bw/day having a significantly higher loss than did the control group.

CLINICAL SIGNS (OFFSPRING)
No data

BODY WEIGHT (OFFSPRING)
The initial litter weight and average pup weight (defined using the live pup litter weight divided by the total number of live pups on day 0) were significantly different only for the 250 mg/kg bw/day group having a significantly increased litter weight and average pup weight compared to that in the control group after adjusting for litter size. The litter size was also a significant variable for these two endpoints with larger litters having litter weights but smaller average pup weights. For the final litter weight and average pup weights, there was also a significant difference, but it was the 1000 mg/kg bw/day group that was significantly different from the control group with both smaller overall litter weights and smaller average pup weight than in the control groups. There were no grossly external abnormalities observed in the pups.


Dose descriptor:
NOAEL
Generation:
F1
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified
Reproductive effects observed:
not specified

Table 2. Summary of Mean Reproductive Parameters for Reproductive Group Female Rats.

 

 

Control (0 mg/kg bw/day)

50 mg/kg bw/day

250 mg/kg bw/day

1000 mg/kg bw/day

Litter Size

Corpora Lutea Counts

Mean

21

 

18

 

17

 

22

 

N/A

Std

5

 

5

 

3

 

5

 

 

N

10

 

8

 

10

 

8

 

 

Total Implants

Mean

15

 

16

 

14

 

13

 

N/A

Std

1.2

 

2.0

 

2.0

 

3.7

 

 

N

10

 

8

 

10

 

8

 

 

Post-Implantation Loss (%)

Mean

9.8

***

4.3

 

8.6

 

23.5

**

N/A

S td

5.9

 

7.7

 

10.2

 

14.3

 

 

N

10

 

8

 

10

 

8

 

 

Days Gestation

Mean

22

 

22

 

22

 

23

***

N/A

Std

1

 

1

 

1

 

1

 

 

N

10

 

8

 

10

 

8

 

 

Total Pups

Mean

13.7

***

16.0

 

13.0

 

10.3

***

N/A

Std

0.8

 

2.3

 

1.8

 

2.8

 

 

N

10

 

8

 

10

 

8

 

 

Total Live Pups Day 0

Mean

13.4

***

15.6

 

12.7

 

9.8

***

N/A

Std

0.8

 

2.8

 

1.7

 

2.7

 

 

N

10

 

8

 

10

 

8

 

 

Male pups

Mean

7

 

8

 

6

 

5

 

***

Std

2

 

3

 

2

 

3

 

 

N

10

 

8

 

10

 

8

 

 

Female pups

Mean

6

 

8

 

6

 

5

 

*

Std

1

 

2

 

2

 

2

 

 

N

10

 

8

 

10

 

8

 

 

Males/Females

Mean

1.4

 

1.1

 

1.2

 

1.2

 

 

Std

0.8

 

0.7

 

0.7

 

0.9

 

 

N

10

 

8

 

10

 

8

 

 

Day 4 Viable pups

Mean

13

***

15

 

13

 

7

***

***

Std

1

 

3

 

2

 

4

 

 

N

10

 

8

 

10

 

8

 

 

Viable/total

Mean

0.97

***

0.96

 

0.97

 

0.74

***

 

Std

0.04

 

0.04

 

0.05

 

0.31

 

 

N

10

 

8

 

10

 

8

 

 

Initial Litter Weight (g) [Live Pups only]

Mean

88

***

100.0

 

93.1

*

64.4

 

 

Std

7.4

 

13.8

 

11.8

 

14.1

 

 

N

10

 

8

 

10

 

8

 

 

Initial Average pup Weight (g) [Live Pups only]

Mean

6.6

***

6.5

 

7.4

***

6.8

 

***

Std

0.5

 

0.6

 

0.4

 

0.7

 

 

N

10

 

8

 

10

 

8

 

 

Final Litter weight (g)

Mean

145.5

***

160.6

 

142.0

 

72.9

***

***

Std

12.6

 

18.8

 

16.0

 

15.5

 

 

N

10

 

8

 

10

 

7

 

 

Final Average pup (g)

Mean

11.0

***

10.5

 

11.3

 

8.9

***

 

Std

0.9

 

1.0

 

1.0

 

1.3

 

 

N

10

 

8

 

10

 

7

 

 

Post-Natal Loss (%)

Mean

0.7

***

1.3

 

0.7

 

24.0

***

*

Std

2.3

 

3.7

 

2.3

 

32.6

 

 

N

10

 

8

 

10

 

8

 

 

Note: Asterisks next to the control indicates a significant treatment effects in the ANOVA, asterisks in the litter column indicates a significant litter effect in the ANOVA with p–values of *<0.05, ** <0.02 or *** <0.01 in the control column and significance in the litter sizes with p-values of *<0.05, **<0.02 or *** <0.01 in the litter significance column. N/A is the litter column indicates that the litter was not used as a covariate in the analysis of that endpoint.

% Post-Implantation Loss = ((# Implantations - # Live at First Litter Check) # Implantations)* 100

% Post-Natal Loss = ((#Live pups Day 0 - # Live pups Day 4) # live pups Day 0)* 100

Conclusions:
The study was conducted according to OECD 422 test guideline, and in compliance with GLP for the registered substance. Based on observations at 1000 mg/kg bw/day (an increase in post-implantation loss, an increase in days of gestation, a decrease in live pups, a decrease in the total viable pups/total, a decrease in final litter weight, a decrease in final average pup weight and an increase in the % of post-natal loss), the NOAEL for reproductive toxicity is 250 mg/kg bw/day.
Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study was conducted according to an appropriate OECD test guideline, and in compliance with GLP.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A key OECD Guideline 422 oral gavage study with dimethoxy(dimethyl)silane (CAS 1112-39-6) is available. Based on observations at 1000 mg/kg bw/day: an increase in post-implantation loss, an increase in days of gestation, a decrease in live pups, a decrease in the total viable pups/total, a decrease in final litter weight, a decrease in final average pup weight and an increase in the % of post-natal loss, the NOAEL for reproductive toxicity is 250 mg/kg bw/day. Based on results observed at 1000 mg/kg bw/day in males: hepatic protoporphyrin accumulation, adrenal cortical atrophy, kidney protein droplet nephropathy, testicular seminiferous tubule degeneration with epididymides involvement and in females rats, periportal vacuolation, the NOAEL for systemic toxicity was also established at 250 mg/kg bw/day.

The testicular seminiferous tubule degeneration was moderate to marked and this can be considered as an adverse effect. However, the implantation rate was not influenced, indicating that the males were still fertile and the dose inducing these effects is at the limit dose. The decreased pup viability and weight can be real developmental toxicity, since periportal vacuolation was the only effect seen at 1000 mg/kg bw/day and no severe maternal toxicity was observed. Therefore, the data is insufficient for classification for fertility effects (Dow Corning Corporation, 2010).

OECD 443: This information will be submitted later based on ECHA decision number CCH-D-2114428794-40-01/F.


Effects on developmental toxicity

Description of key information

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422), rat:

NOAEL(developmental toxicity) = 250 mg/kg bw/day
NOAEL(maternal toxicity) = 250 mg/kg bw/day

Prenatal developmental toxicity study (OECD TG 414), rat:

NOAEL (maternal toxicity) < 100 mg/kg bw/day

NOAEL (developmental toxicity) = 100 mg/kg bw/day

In order to fulfil the standard information requirements, a GLP-compliant prenatal developmental toxicity study in rabbits via the oral route following OECD TG 414 is proposed according to Annex X, Column 1, Section 8.7.2.

Link to relevant study records
Reference
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
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
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
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
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
low dose (LD)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
medium dose (MD)
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.
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.
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.
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.
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
Key result
Abnormalities:
no effects observed
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.
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
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: decreased fetal weight
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
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.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable studies, and is thus sufficient to fulfil the standard information requirements set out in Annex IX, 8.7, of Regulation (EC) No. 1907/2006.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Reliable developmental toxicity data are available from a screening study (OECD 422) and from a prenatal developmental toxicity study (OECD 414) conducted with the submission substance.

An OECD Guideline 422 oral gavage study with dimethoxy(dimethyl)silane (CAS 1112-39-6) is available (Dow, 2010). Based on observations at 1000 mg/kg bw/day: an increase in post-implantation loss, an increase in days of gestation, a decrease in live pups, a decrease in the total viable pups/ total, a decrease in final litter weight, a decrease in final average pup weight and an increase in the % of post-natal loss, the NOAEL for developmental toxicity is 250 mg/kg bw/day (Domoradzki, J.Y.2010). Based on results observed at 1000 mg/kg bw/day in males: hepatic protoporphyrin accumulation, adrenal cortical atrophy, kidney protein droplet nephropathy, testicular seminiferous tubule degeneration with epididymides involvement and in females rats, periportal vacuolation, the NOAEL for systemic toxicity was also established at 250 mg/kg bw/day.

The testicular seminiferous tubule degeneration was moderate to marked and this can be considered as an adverse effect. However, the implantation rate was not influenced, indicating that the males were still fertile and the dose inducing these effects is at the limit dose. The decreased pup viability and weight can be real developmental toxicity, since periportal vacuolation was the only effect seen at 1000 mg/kg bw/day and no severe maternal toxicity was observed. Therefore, the data is insufficient for classification and no histopathology was performed on the pups.

In a prenatal developmental toxicity study according to OECD TG 414 and in compliance with GLP, nulliparous and non-pregnant female Wistar rats were mated with males (2:1 ratio) and divided into four groups of each 23-24 animals (BSL, 2021, Draft amended report). The pregnant females were administered the test substance dimethoxy(dimethyl)silane via gavage from gestation day 5 to 19 at dose levels of 100 (LD), 300 (MD) and 1000 mg/kg bw/day (HD), respectively. The animals of the control group received the vehicle dried and de-acidified corn oil.

Maternal findings

One female in HD group was moribund sacrificed due to animal welfare reason on GD 15 and showed piloerection, pale skin and scratch/cut on the day of sacrifice. There were no changes in body weight and food consumption during the gestation period and were to be comparable to control. At macroscopic examination, the animal was observed with enlarged heart, pale colored lungs, stomach, duodenum, jejunum, ileum, cecum, colon, liver and kidneys, a white spotted and enlarged spleen and a mass (dark, flexible, 2 cm) on the left hind limb. 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.

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. Moving the bedding and increased salivation observed in few animals of the test item-treated groups were noted in direct timely relation to dose administration or in anticipation thereof and thus were considered to be a sign of discomfort or a local reaction.

There was a moderate effect on body weight development for the HD group after initiation of treatment which was considered an adverse effect of treatment with the test item.After initiation of treatment, body weight gain was lower during GD 5-8 in the MD and HD group. From gestation day GD 11, mean body weight gain remained lower in all dose groups with statistical significance in the HD group during gestation days 14-17. Body weight gain was dose-dependently lower in dose groups and statistically significantly lower in the HD group when related to the entire study period (GD 0-20). From GD 8 onwards body weights of the HD group were slightly lower with the greatest effect on GD 20 without statistical significance. However, this effect on body weight development in HD group was assumed test item related. Food consumption was lower in females of the dose groups before initiation and tended to decrease dose-dependently in correlation to lower body weight gain after initiation of treatment. Food consumption was lower from GD 5-8 and gestation days 8-11 and resulted in statistically significantly lower food consumption of the HD group from gestation days 5-8 and 11-14.

Successful mating resulted in 19/23 pregnancies in the control group, 22/24 pregnancies in the LD group, 20/24 pregnancies in the MD group and 19/24 pregnancies in the HD group. Maternal weight and adjusted maternal weight showed a slight dose-dependent trend towards lower weight in dose groups on the day of caesarean section. This effect was consistent with the body weight development during the gestation phase and it is considered to be test item related . Uterine weight was slightly lower in all dose groups without dose dependency. Statistically significantly higher incidence of late resorptions was observed in the HD group; however these values are within historical control data and not considered to be treatment related effects.

There were no biologically relevant or statistically significant effects on prenatal data. Prenatal parameters like number of corpora lutea, implantation sites, live foetuses and early resorptions, pre- and post-implantation loss and sex ratio remained unaffected in the dose groups when compared to the control group. Slight differences between dose groups and the controls followed no dose-dependency and thus were considered not toxicologically relevant. No dead foetuses were observed in any of the test item-treated groups or the control group.

Thyroid/parathyroid weight showed no statistically significant or biologically relevant effects when comparing dose groups to the control group. Weights were comparable between the groups. At histopathological evaluations,thyroid follicular cell hypertrophy was observedfrom 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 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 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. However, additional data (e.g. evaluation of liver) may be needed to fully elucidate the pathogenesis underlying the mentioned thyroid gland change.

A statistically significantly higher mean T3 level was observed in the HD group compared to controls. The mean TSH level was moderately higher in the HD group compared to the control group. T4 levels showed no relevant differences between dose groups and the control group. However, there was no dose dependency in all three hormones analysed and all values were with in historical control range; hence the effects of the test item on hormone levels were considered not toxicologically relevant.

There were no treatment-related macroscopic pathological lesions observed in any of the dams at necropsy.

Foetal Findings

The mean foetus weight was found to be lower in the HD group which was considered test item-related. Independent of the gender, foetus weight was statistically significantly lower in the HD group compared to the control group. Also in each of the genders, foetus weight was statistically significantly lower in the MD and HD group compared to the corresponding controls, including statistically significant lower weight of LD male foetus weight. These are 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. There was no test-item related effect observed on AGD in any of the groups.Testicular (abdominal) descent was observed to be complete in all examined male foetuses and no test item related effects were observed.

At skeletal examination, a trend towards incomplete ossification of several bones could be observed with statistically significantly higher foetal incidences of incomplete ossification in the HD group. These include bones of the skull, clavicula, sternebra, ribs, pelvis, vertebra (dumbbell, bipartite, irregular, hemicentric and asymmetric ossification), forelimbs and hindlimbs. Foetal incidence for missing ossification was statistically significantly higher mostly in the HD group for forelimb metacarpals, hindlimb metatarsals, cervical centra, thoracic centra, caudal centra and caudal arches.

Increasing incidence of misshapen/bent/thick bones was observed in dose groups resulting in statistically significant differences of some findings which were considered test item-related. Related to this, misshapen humeri, ulnae, radii, femora, tibiae, fibulae and bent ischia and fibulae showed statistically significantly higher foetal incidence in the HD group. Bent or thick bones are considered as developmental delays that will be corrected after birth [Kimmel et al., 2014].Also, broken fibulae were identified. As the broken shafts were adjacent to each other they are likely to heal during the lactation period.

Misshapen bent scapulae and bent scapular spines were observed with a slight dose dependent trend resulting in a statistically significantly higher foetal incidence in the HD group. The finding of bent scapula appears to be transient and completely repaired post-natally. Without corresponding external observations of short and/or malformed limbs they maybe classified as variation[Kimmel et al., 2014].

Wavy ribs were observed with statistically significantly higher foetal incidence in the MD and HD group. In general, wavy ribs are typically classified as transient and reversible post-natally and thus may be considered as variations [Kimmel et al., 2014]which are not consideredas an adverse effect of treatment with the test item. However, they are reported to occur more often in the presence of maternal and/or foetal toxicity, secondary to generalized toxicity or stress[Kimmel et al., 2014]. Thus, the significantly higher incidence of wavy ribs in the HD group might be related to maternal toxicity with the observed reduced body weight and food consumption of dams of the HD group.

The finding of split thoracic centra and wide intersternebral cartilage which was observed with a statistically significantly higher foetal incidence in the HD group was considered as variations [DeSesso and Scialli, 2018]. This observation indicates the delay in ossification and development of sternum which will normalise at post natal development. Split and dumbbell-shaped cartilaginous vertebral centra have been shown to normalise largely in rats treated with ethylene glycol during GD 6-15 by 63 days postnatally [Marr et al., 1992].

The presence of bilateral supernumerary (14th) thoracolumbal full ribs 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. Cervical ribs were noted in few foetuses throughout all dose groups with a slight dose-dependent trend towards higher incidence of cervical ribs with increasing dose of test item. In general, supernumerary ribs are skeletal alterations that can occasionally be seen in animals of this strain and age.Disruption of the cervical vertebral pattern is expected to be more harmful than disruptions at the thoracic or lumbar level, because thecaudal regions of the vertebral column develop later, during less vulnerable and interactive stages [Schut et al., 2020] andIn 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). 

Foetal incidence of pelvic girdle caudal shift increased dose-dependently and resulted in statistically significantly higher incidences in the HD group. However, 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 and are not considered adverse.

The HD group was observed with a statistically significantly higher foetal incidence of supernumerary cervical cartilaginous ribs and a statistically significantly higher foetal incidence of cervical cartilaginous ribs fused to the first costal cartilage, according to the observed tendency of cervical ribs to fuse, sometimes to the first rib[Schut et al., 2020]. Both findings were considered test item-related. These cartilaginous structures may undergo skeletal remodelling postnatally and thus be regarded as variations [DeSesso and Scialli, 2018]

The finding of wide and split intersternebral cartilage showed a statistically significantly higher foetal incidence in the HD group. The appearance of wide intersternebral cartilage may be considered a variation[DeSesso and Scialli, 2018]. Split intersternebral cartilage may be considered as a delay in fusion of sternal bars [DeSesso and Scialli, 2018]and thus as a variation. The sterna of rodents and humans undergo most of their ossification postnatally [DeSesso and Scialli, 2018]. Foetal incidence of supernumerary costal cartilage was dose-dependently higher in dose groups resulting in statistically significantly higher foetal incidence in the HD group. The appearance of showed a trend towards higher incidence in dose groups resulting in statistical significance in the HD group.

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.

 

Thus, 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.

 

 

References:

DeSesso JM and Scialli AR. Bone development in laboratory mammals unsed in developmentaltoxicity studies. Birth Defects Res 2018; 110:1157-1187. doi: 10.1002/bdr2.1350

Guidance on Evaluation of Reproductive Toxicity Data, Monograph No. 31, Brussels, February 2002

Kimmel et al (2014). Relationship between bent long bones, bent scapulae and wavy ribs: malformations or variations? Birth Defects Research 101

Marr MC, Price CJ, Myers CB, Morrissey RE (1992) Developmental stages of the CD (Sprague-Dawley) rat skeleton after maternal exposure to ethylene glycol. Teratology 46:169-181

Schut et al., 2020,Exploring copy number variants in deceased fetuses and neonates with abnormal vertebral patterns and cervical ribs;Birth Defects Research,112,1513–1525

 





Justification for classification or non-classification

The available data on developmental toxicity do not meet the crieteria for classification according to Regulation (EC) No 1272/2008.

However, the available data on toxicity to reproduction of the registered substance is not sufficient for classification according to Regulation (EC) 1272/2008. Therefore, no conclusion can be made on the classfication for reproductive toxicity until the results of the OECD 443 are available.

No information is available on effects via lactation currently.

Additional information