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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:
18th of December 2019 to 12th of June 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report Date:
2020

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
July 2018
Deviations:
yes
Remarks:
The measured concentration of the first formulation prepared at 12.5 mg/ml for the 50 mg/kg bw/day group was -18.4% of nominal, equating to an approximate dose of 41 mg/kg bw/day. This formulation was administered for up to seven days (GD 6-13).
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Remarks:
Clear, colourless

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl: CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd
- Age at study initiation: 70 to 76 days.
- Weight at study initiation: 214 to 262 grams
- Fasting period before study: not specified
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid. Solid (polycarbonate) bottom cages were used during the acclimatisation and gestation periods. Grid bottomed cages were used during pairing. Cages were suspended above absorbent paper which was changed daily during pairing. Solid bottom cages contained softwood based bark-free fiber bedding which was changed at appropriate intervals each week. During the acclimatisation, up to four animals were caged together. During pairing, one (stock) male and one female were together and during gestation, one female was caged.
- Diet: SDS VRF1 Certified pelleted diet, ad libitum. The diet did not contain any added antibiotic or other chemotherapeutic or prophylactic agent. A soft white untreated Aspen chew block was provided to each cage throughout the study and replaced when necessary.
- Water: water was supplied from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals, ad libitum.
- Acclimation period: 7 days before commencement of pairing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 to 25°C
- Humidity (%): 40-70%
- Air changes (per hr): at least 15 air changes / hour. Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

IN-LIFE DATES: Not specified

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
Dried and de-acidified corn oil (50% silica gel desiccant, 25% aluminium oxide neutral and 25% aluminium activated acidic (40 grams, 20 grams and 20 grams respectively per litre of oil).
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: A series of formulations at the required concentrations were prepared by dilution of individual weightings of the test item in ascending order of concentration. The humidity within the glove box, used for all formulation procedures, was checked to ensure it was 10% or below. The required amount of test item was added to the required volume of vehicle. Each formulation was stirred using a magnetic stirrer until uniformly mixed and then transferred to final glass containers, via syringe, whilst magnetically stirring.
Each glass container of formulation was filled to reduce the volume of air (head space) and was then purged with nitrogen. Formulations were issued daily and were issued in a cool box containing ice although, the dose containers did not come into contact with the ice.The formulations were prepared weekly and stored refrigerated (2 to 8°C).

VEHICLE
- Justification for use and choice of vehicle: The vehicle, dried and de-acidified corn oil, was selected in conjunction with the sponsor and according to the appropriate OECD guideline.
- Concentration in vehicle: Nominal concentrations: 0, 12.5, 62.5 and 187.5 mg/mL. Formulated concentrations: 0, 16.5, 82.5 and 247.5 μL/mL.
- Amount of vehicle: 4 mL/kg
- Purity: Not specified
- Preparation of vehicle: Dried and de-acidified corn oil was mixed and placed in a vented oven set to 250ºC overnight or for at least eight hours and then allowed to dry in a dessicator. The oil and dry materials were mixed in a suitable container, using a stir bar on a magnetic stirring plate, for at least two hours under a nitrogen purge. The oil and dry materials mix was allowed to settle for a minimum of 30 minutes. The oil was filtered with 0.22 μm cellulose acetate filter system with vacuum to remove the alumina and silica. The filtered dried and de-acidified corn oil was kept in amber glass bottles, topped with a nitrogen purge, sealed with parafilm and stored with desiccant material at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The homogeneity and stability of formulations during storage were confirmed as part of another study. Samples of each of the first and last preparation formulations were analysed for achieved concentration of the test item.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused: A female was taken to a male's cage
- M/F ratio per cage: 1:1 with identified stock males
- Length of cohabitation: until mating was confirmed.
- In case of unsuccessful mating, the female was taken back to its original cage and mating of the female with another male or at another time was considered
- Verification of same strain and source of both sexes: Yes
- Proof of pregnancy: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm referred to as day 0 of pregnancy. Only females showing at least two copulation plugs were allocated.
- Any other deviations from standard protocol: No
Duration of treatment / exposure:
Between GD 6 to 19
Frequency of treatment:
Daily
Duration of test:
20 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
nominal concentration: 0 mg/mL
Dose / conc.:
50 mg/kg bw/day
Remarks:
nominal concentration: 12.5 mg/mL
Dose / conc.:
250 mg/kg bw/day
Remarks:
nominal concentration: 62.5 mg/mL
Dose / conc.:
750 mg/kg bw/day
Remarks:
nominal concentration: 187.5 mg/mL
No. of animals per sex per dose:
90 females, 20 animals per group. Spare animals were removed from the study room after treatment commenced.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels for this study (0, 50, 250 and 750 mg/kg bw/day) were selected in conjunction with the Sponsor and based on the results from the preliminary dose range finding study (Covance Laboratories, 2020). In the study pregnant female rats were given daily oral (gavage) administration of 1,1,3,3-tetramethyldisiloxane in corn oil at doses of 0, 300, 600 and 1000 mg/kg bw/day.
- At 1000 mg/kg bw/day overall body weight gain and gravid uterine weight were lower compared to control. The numbers of pre- and post-implantation losses were higher and resulted in a low mean litter size at 1000 mg/kg bw/day compared to control. Pre-implantation loss was higher at 600 mg/kg/day compared to control also. Liver weights appeared to be marginally higher at 600 or 1000 mg/kg bw/day compared to control; however, no definite relationship to treatment was inferred. The majority of foetuses in one litter at 600 mg/kg bw/day and some or the majority of litters at 1000 mg/kg bw/day showed the major foetal abnormality of domed craniums. At 1000 mg/kg/day, the majority of foetuses in one litter and one foetus from each of two litters had whole body oedema and protruding tongue or short snout was evident in one or two foetuses, respectively.
- Rationale for animal assignment: randomised

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The health conditions of the animals were recorded at least twice daily, preferably at the same time each day. Once daily all animals were observed for morbidity and mortality. Signs associated with dosing were also recorded daily.

DETAILED CLINICAL OBSERVATIONS: Yes
A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.

BODY WEIGHT: Yes
The weight of each adult was recorded on Days 0, 3 and 6-20 after mating.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 after mating inclusive.

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 after mating.
- Organs examined: The kidneys, liver and thyroid gland were weighed. Gravid uterine weight was also recorded. The kidneys, liver, thyroid gland, and uterus were fixed at necropsy. The thyroid gland was examined at histopathology.

OTHER: Thyroid hormone analysis was performed prior to sacrifice. Blood samples were collected from all animals prior to termination. The animals were not fasted overnight. The samples were analysed for T3, T4 and TSH levels.
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
- Other: The number of uterine implantation sites were checked after staining with ammonium sulphide
Fetal examinations:
- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, all per litter
- Skeletal examinations: Yes, all per litter
- Head examinations: Yes, all per litter
Statistics:
A parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. For pre-treatment data, analysis of variance was used to test for any group differences. When significant, inter group comparisons using t-tests with the error mean square from the one-way analysis of variance were made. For all other analyses, the F1 approximate test was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test was performed. A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For pre-treatment data, Kruskal-Wallis’ test was used to test for any group differences. Where this was significant (p<0.05) intergroup comparisons using Wilcoxon rank sum tests were made. For all other analyses the H1 approximate test, the non parametric equivalent of the F1 test described above was applied. If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test was performed instead.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Two animals receiving 250 mg/kg bw/day and four animals receiving 750 mg/kg bw/day showed transient salivation very soon after dosing, during days 17-19 of gestation. This was considered attributed to the palatability of the test item formulations and was therefore, not considered to be toxicologically significant. There were no signs attributable to dosing at 50 mg/kg bw/day.
There were no clinical observations attributable to treatment at 50, 250 or 750 mg/kg bw/day.
One animal receiving 750 mg/kg bw/day was observed with a swollen area on the lower ventral surface on the day of termination that was determine as a firm inguinal mammary mass at necropsy.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality was observed.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The group mean body weight performance and overall gain (GD 6-20) of females treated with tetramethyldisiloxane at 50, 250 or 750 mg/kg bw/day were comparable to that of the control group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Group mean food consumption of females treated with 50, 250, or 750 mg/kg bw/day tetramethyldisiloxane was similar to the controls during gestation Days 6-20.
Food efficiency:
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):
Group mean adjusted kidney and liver weights were marginally higher in females treated with 750 mg/kg bw/day (111% and 112% of control, respectively). However, the difference from control was marginal and there was no macroscopic finding in either tissue and therefore, not considered as treatment related. Group mean kidney and liver weights at 50 or 250 mg/kg bw/day and thyroid weight at 50, 250 or 750 mg/kg bw/day were unaffected by treatment compared to controls.

The mean gravid uterine weight for females treated with 1,1,3,3-tetramethyldisiloxane was comparable to the mean gravid uterine weight from the control females, as was adjusted body weight and body weight gain.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The macroscopic examination performed following treatment on GD 6 to 19 revealed no treatment related findings.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Minimal follicular cell hypertrophy in the thyroid glands was seen in five females treated with 750 mg/kg bw/day and in one female from the control group. All other histological changes were considered to be unrelated to treatment.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
At 750 mg/kg bw/day, there was a suggestion of a non-adverse effect on thyroid function (Table 1). Mean serum T4 and T3 concentrations were slightly but statistically significantly lower than those in the controls (no dose response was apparent) and microscopic examination of the thyroids revealed five females with follicular cell hypertrophy compared to one in the control. Individual T4 and T3 concentrations were highly variable and all T4 concentrations were within the concurrent control range. The reduction in T4/T3 concentrations was only slight and there was no obvious correlation to the occurrence of follicular cell hypertrophy and mean serum TSH concentrations were unaffected, which would have been expected to accompany reduced T4/T3 concentrations and result in thyroid follicular cell hypertrophy. Therefore, no definite effect of treatment at 750 mg/kg bw/day on thyroid function is inferred. At 50 or 250 mg/kg bw/day, the mean serum T4/T3 concentrations were slightly statistically significantly lower than the control group. Although, no dose response, supporting microscopic changes in the thyroids or increase in TSH concentrations occurred. Therefore, the slightly reduced T4/T3 concentrations are considered to be incidental and not related to treatment.

Maternal developmental toxicity

Number of abortions:
not specified
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The numbers of pre- and post-implantation loss were unaffected by the treatment in all dose groups.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
The numbers of resorptions were unaffected by treatment at 50, 250 or 750 mg/kg bw/day.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The number of early or late resorptions were unaffected by the treatment in all dose groups.
Dead fetuses:
no effects observed
Description (incidence and severity):
The number of live foetuses were unaffected by the treatment in all dose groups.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
One animal that received 250 mg/kg bw/day was not pregnant. Therefore, reproductive assessment was assessed using 20 control animals and 20, 19 or 20 animals treated at 50, 250 or 750 mg/kg bw/day, respectively.
Other effects:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Overall foetal weight (male and female combined) was statistically significantly lower at 250 or 750 mg/kg bw/day (91% or 80% of control, respectively) and the resultant total litter weight at 750 mg/kg bw/day was statistically significantly lower (84% of control). Foetal and litter weights were unaffected by treatment at 50 mg/kg bw/day compared to control.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There was no reduction in the number of live offspring
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex ratio of males to females was unaffected by the treatment in all dose groups.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
Overall foetal weight (male and female combined) was statistically significantly lower at 250 or 750 mg/kg bw/day (91% or 80% of control, respectively) and the resultant total litter weight at 750 mg/kg bw/day was statistically significantly lower (84% of control). Foetal and litter weights were unaffected by treatment at 50 mg/kg bw/day compared to control.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
When compared with control, there was an increase in incidence of thoracic vertebral abnormalities; medially thickened/kinked ribs; short supernumerary cervical ribs; short and full supernumerary 14th ribs; 20 thoracolumbar vertebrae; unilateral caudal shift of the pelvic girdle; a generalised delay in ossification (including cranial bones, sternebrae, all vertebrae, ribs, pelvic and digit bones) and domed cranium. All parameters exceeded the historical control data (HCD) range.

There was an increase in incidence of some of the abnormalities/variants, when compared with control, at 250 mg/kg bw/day, but to a lesser degree. These included short supernumerary 14th rib; incomplete ossification of cranial bones, sternebrae, thoracic and sacral/caudal vertebrae and pelvic bones. With the exception of incompletely ossified cranial bones, all exceeded the HCD range.

Many of these abnormalities and/or variants may be associated with the lower foetal weight observed at 750 mg/kg bw/day and to a lesser extent at 250 mg/kg bw/day. Incomplete ossification and slightly domed cranium are transient stages in foetal development and indicative of foetal immaturity.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
There was an increase in abnormalities affecting the heart and major blood vessels at 750 mg/kg bw/day compared to control. These included double/retroesophageal/right sided aortic arch; retroesophageal subclavian artery; dilated ascending aorta/pulmonary trunk and muscular/membranous ventricular septal defect. Minor blood vessels were also affected, with variation in the origin of the subclavian, carotid and innominate arteries from the aortic arch.
Many of these abnormalities and/or variants may be associated with the lower foetal weight observed at 750 mg/kg bw/day and to a lesser extent at 250 mg/kg bw/day. Partially undescended lobe of thymus, small renal papilla, shiny skin in foetal development and indicative of foetal immaturity. With the exception of partially undescended thymus, all exceeded the HCD range.
Other effects:
no effects observed
Description (incidence and severity):
Anogenital distance was unaffected by maternal treatment at all dose groups.

Effect levels (fetuses)

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
skeletal malformations
visceral malformations

Fetal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: skull
skeletal: rib
skeletal: vertebra
skeletal: pelvic girdle
visceral/soft tissue: cardiovascular
Description (incidence and severity):
Skeletal: an increase in incidence of thoracic vertebral abnormalities; medially thickened/kinked ribs; short supernumerary cervical ribs; short and full supernumerary 14th ribs; 20 thoracolumbar vertebrae; unilateral caudal shift of the pelvic girdle; a generalised delay in ossification (including cranial bones, sternebrae, all vertebrae, ribs, pelvic and digit bones) and domed cranium.

Visceral: abnormalities affecting the heart and major blood vessels. These included double/retroesophageal/right sided aortic arch; retroesophageal subclavian artery; dilated ascending aorta/pulmonary trunk and muscular/membranous ventricular septal defect. Minor blood vessels were also affected, with variation in the origin of the subclavian, carotid and innominate arteries from the aortic arch.

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
750 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Table 1 - Summary of adult thyroid hormone results on Day 20 of gestation

Group

1

2

3

4

Dose (mg/kg bw/day)

0

50

250

750

Triiodothyronine (pg/mL)

564

473**

482**

479**

Thyroxine (pg/mL)

19300

16000**

16000**

15600**

Thyroid Stimulating Hormone (pg/mL)

 

1530

 

1210

 

1430

 

1770

** p<0.01

Table 2 -Summary of treatment related findings in the thyroids for animals killed following treatment on Days 6 to 19 of gestation

Group/sex

1F

2F

3F

4F

Dose (mg/kg/bw/day)

0

50

250

750

Hypertrophy, Follicular Cell

 

 

 

 

Minimal

1

0

0

5

Total

1

0

0

5

Number of tissues examined

20

20

20

20

Applicant's summary and conclusion

Conclusions:
In a prenatal developmental toxicity study in rats for 1,1,3,3-tetramethyldisiloxane, conducted according to OECD Test Guideline 414 and in compliance with GLP, the reported NOAEL for maternal toxicity was 750 mg/kg bw/day, the highest dose tested. The NOAEL for developmental toxicity was concluded to be 250 mg/kg bw/day based on abnormalities observed at 750 mg/kg/day.