[3aR-(3aα,5aβ,9aα,9bβ)]-dodecahydro-3a,6,6,9a-tetramethylnaphtho[2,1-b]furan

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 23 March 2021 to 07 July 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD 414 Guideline without deviations.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

other: Draft report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

1

Reference substance name:

(3aS,5aR,9aR,9bS)-3a,6,6,9a-Tetramethyldodecahydronaphtho[2,1-b]furan

Cas Number:

234431-64-2

Molecular formula:

C16H28O

IUPAC Name:

(3aS,5aR,9aR,9bS)-3a,6,6,9a-Tetramethyldodecahydronaphtho[2,1-b]furan

2

Reference substance name:

[3aR-(3aα,5aβ,9aα,9bβ)]-dodecahydro-3a,6,6,9a-tetramethylnaphtho[2,1-b]furan

EC Number:

229-861-2

EC Name:

[3aR-(3aα,5aβ,9aα,9bβ)]-dodecahydro-3a,6,6,9a-tetramethylnaphtho[2,1-b]furan

Cas Number:

6790-58-5

Molecular formula:

C16H28O

IUPAC Name:

(3aR,5aS,9aS,9bR)-3a,6,6,9a-tetramethyl-dodecahydronaphtho[2,1-b]furan

Test material form:

solid

Remarks:

white solid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

RccHan®:WIST

Details on test animals or test system and environmental conditions:

RATIONALE FOR ANIMAL MODEL:
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Han Wistar rat strain was used because of the historical control data available at this laboratory.

TEST ANIMALS
- Source: Envigo RMS Limited.
- Age at study initiation: 78 to 84 days old.
- Weight at study initiation: 169 to 219 g
- Housing: Acclimatization - up to four animals; During pairing - one (stock) male and one female; Gestation - one female
Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization 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.
- Diet: SDS VRF1 Certified pelleted diet, ad libitum
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: Six days before pairing.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod: Artificial lighting, 12 h light : 12 h dark
Environmental Enrichment
- Aspen chew block: A soft white untreated wood block; provided to each cage throughout the study and replaced when necessary.
- Plastic shelter: Provided to each cage throughout the study (except during pairing) and replaced at the same time as the cages.

IN-LIFE DATES: From: 31 March 2021 To: 30 April 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

RATIONALE FOR ROUTE OF ADMINISTRATION:
The oral gavage route of administration was chosen to simulate the conditions of possible human exposure.

PREPARATION OF DOSING SOLUTIONS:
Method of preparation: The required amount of test item was ground in a mortar using a pestle and mixed with some vehicle to form a paste. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogenizer.
A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.
Frequency of preparation: Weekly.
Storage of formulation: Refrigerated (2-8 °C)

VEHICLE
- Concentration in vehicle: 25, 100 and 200 mg/mL
- Dose volume: 4 mL/kg

STABILITY AND HOMOGENEITY
Before commencement of treatment, the suitability of the proposed mixing procedures was determined as part of another study, Covance GLP Study 8449087. In that study formulations in the range 1 and 200 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix. Stability was determined as 15 days refrigerated (2 to 8°C) and for one day at ambient temperature (15 to 25°C).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Achieved concentration: Samples of each formulation prepared for administration in first and last week of treatment were analyzed for achieved concentration of the test item.

Details on mating procedure:

- M/F ratio per cage: 1:1 with identified stock males
- Daily checks for evidence of mating: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm.
- Day 0 of gestation: When positive evidence of mating was detected.

A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.

Duration of treatment / exposure:

Females were treated from Day 6 to Day 19 (inclusive) after mating.

Frequency of treatment:

Once daily at approximately the same time each day.

Duration of test:

From mating (Day 0) to necropsy (Day 20).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Test item

Doses / concentrations 3

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

Test item

Doses / concentrations 4

Dose / conc.:

800 mg/kg bw/day (nominal)

Remarks:

Test item

No. of animals per sex per dose:

20 mated females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses used in this study (0, 100, 400 or 800 mg/kg/day) were selected in conjunction with the Sponsor. A combined repeat dose toxicity study with reproductive/developmental toxicity screening test (OECD 422) was conducted with the test substance in 2008/2009 (Harlan study no C05606, Füllinsdorf, Switzerland) using Han Wistar rats. In that study, the test substance was administered to rats (n = 10/group) at doses of 0, 100, 400 or 800 mg/kg/day in corn oil by oral gavage. The general NOAEL and NOEL from that study were determined to be 800 and 100 mg/kg/day, respectively. The developmental NOAEL and NOEL were determined to be 800 and 400 mg/kg/day, respectively. Based on the observations from that study it would be anticipated that animals dosed using a limit dose approach (i.e., high dose of 1000 mg/g/day) would be expected to exhibit adverse effects that would not be consistent with animal welfare testing requirements. Therefore, dose levels of 0, 100, 400, and 800 mg/kg/day were selected for use on this OECD 414 study.

- Rationale for animal assignment: On the day of positive evidence of mating (Day 0). Only females showing at least two copulation plugs were allocated.
Method: To group and cage position in the sequence of mating. Females mating on any one day were evenly distributed amongst the groups. Allocation was controlled to prevent any stock male from providing more than one mated female in each treatment group.

Examinations

Maternal examinations:

MORTALITY: Yes
- A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed observations were recorded daily at the following times in relation to dose administration: A pre-dose observation; One to two hours after completion of dosing and as late as possible in the working day.
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
- Time schedule for examinations: The weight of each adult was recorded on Days 0, 3 and 6-20 after mating.

FOOD CONSUMPTION: 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, 6-7, 8-10, 11-13, 14-16 and 17-19 after mating inclusive.

POST-MORTEM EXAMINATIONS: Yes
- Animals surviving until the end of the scheduled study period were killed on Day 20 after mating by Carbon dioxide asphyxiation.
- Necropsy: All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in 10% Neutral Buffered Formalin. Terminal blood samples were also required for thyroid hormone analysis.
- Organ weights: the organs weighed, tissue samples fixed and sections examined microscopically are detailed in Table 7.8.2/1.
For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled intervals.
- Histology: For all adult females, tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
- Light microscopy: Thyroid was preserved for examination for premature deaths and terminal sacrifice of all animals. Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.

Ovaries and uterine content:

For females surviving to term, the ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight (including cervix and ovaries): Yes
- Number of corpora lutea: Yes
- Number of implantation sites: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of Fetuses (live and dead): Yes

In apparently non pregnant animals and for apparently empty uterine horns, the number of uterine implantation sites were checked after staining with ammonium sulphide.

Blood sampling:

THYROID HORMONE ANALYSIS: Yes
- Blood samples (1.0 mL) were collected at termination of the study in all surviving adults in sublingual vein after an isoflurane anaesthesia. Samples were kept at ambient temperature (15 to 25°C) for a minimum of 30 minutes prior to centrifugation (2000 g for 10-min at 4°C).
- Number of aliquots: Two per animal. Aliquot 1: 0.2 mL serum for T3/T4; Aliquot 2: residual serum for TSH.

Fetal examinations:

FETAL EXAMINATION & PROCESSING
- Method of kill for fetuses: Chilling on a cool plate (approximately 0 °C)
- Examination of all viable fetuses and placentae: Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded and particular attention was paid to external genital organs of male fetuses. The sex and ano genital distance of each fetus was recorded.
- Examination of nominally 50% of fetuses in each litter: Sexed internally and eviscerated.
- Fixation: Fetuses eviscerated were fixed in Industrial Methylated Spirit (IMS). Remaining fetuses were fixed whole in Bouin’s fluid.
- Processing: Bouin’s fixed fetuses were subject to free-hand serial sectioning.
IMS fixed fetuses were processed and stained with Alizarin Red.

FETAL PATHOLOGY EXAMINATION
- Bouin’s fixed fetuses: Serial sections were examined for visceral abnormalities.
- Alizarin Red stained fetuses: Assessed for skeletal development and abnormalities.

Statistics:

See " Any other information on materials and methods incl. tables"

Indices:

Prenatal losses are separated into pre- and post-implantation phases. Pre-implantation loss
was considered to reflect losses due to non-fertilization of ova and failure to implant. It was
calculated from the formula:
Pre-implantation loss (%) = [(Number of corpora lutea – Number of implantations) / Number of corpora lutea] x 100

Where the number of implantations exceeded the number of corpora lutea observed,
pre-implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to
have occurred).
Post-implantation loss was calculated from the formula:
Post-implantation loss (%)= [(Number of implantations – Number of live fetuses) / Number of implantations] x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At routine physical examination three females receiving 800 mg/kg/day were considered to have a thin build; there were no other signs that were considered to relate to treatment.
At 400 or 800 mg/kg/day an increased incidence of piloerection was observed in association with dose administration.
In addition, chin rubbing and increased salivation was observed for a few animals receiving 400 or 800 mg/kg/day; this is commonly observed in oral gavage studies and is considered to relate to palatability of the formulation, rather than a direct effect of the test item and is therefore considered to be of no toxicological significance.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female (no. 74) receiving 800 mg/kg/day was killed for welfare reasons on GD7 due to a swollen, dark hindlimb and loss of locomotion. Macroscopic examination revealed tibiotarsal trauma. This death was accidental and unrelated to administration of ST 10 C 08.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Absolute body weights were statistically significantly low when compared with Controls for females receiving 800 mg/kg/day from GD8 to GD10, and on GD19 and GD20. Overall body weight gain at 800 mg/kg/day was low at approximately 84% of Controls; however, this difference did not attain statistical significance. Body weight gain at 100 or 400 mg/kg/day was unaffected by treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

From GD6 to GD9 females receiving 800 mg/kg/day showed low consumption when compared with Controls (p<0.01); subsequent consumption was similar or slightly higher than Controls. At 400 mg/kg/day there were periods of alightly high consumption late gestation, but overall food consumption was unaffected by treatment.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Mean serum T3 and T4 concentrations in animals treated with ST 10 C 08 were not statistically significant different from mean concurrent Control values.
Administration by oral gavage of ST 10 C 08 at 800 mg/kg/day had a statistically significant effect on serum TSH levels in female rats when compared to the concurrent control group (p<0.01); circulating levels at 100 or 400 mg/kg/day were similar to Controls.

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Animals that received 400 or 800 mg/kg/day showed high mean thyroid weight at approximately 125% (p<0.05) and 142 % (p<0.01) of Controls, respectively. this correlated with the microscopic finding of follicular hypertrophy in some animals.
The gravid uterine weight was statistically significantly low when compared with Controls for females that received 800 mg/kg/day (p<0.01). The maternal body weight gain, following adjustment for the gravid uterine weight, showed no adverse effect of treatment at dose levels up to and including 800 mg/kg/day.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no maternal macroscopic abnormalities detected at scheduled termination that were considered to relate to administration of ST 10 C 08.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At terminal sacrifice, thyroid follicular cell hypertrophy (minimal severity) was seen in eight females administered 800 mg/kg/day ST 10 C 08 and in one female administered 400 mg/kg/day ST 10 C 08.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

See table of results in the section "Attached background material".

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

There was no evidence of an effect of maternal treatment on the mean number of implantations or sex ratio at any dose level investigated.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

The mean number of early resorptions were slightly high at 800 mg/kg/day and when compared with Controls the resultant post-implantation loss was high (p<0.05) and the live litter size was low (p<0.05). At 100 or 400 mg/kg/day the mean number of resorptions, the extent of the pre- and post- implantation loss and total number of live young were similar Controls and considered to be unaffected by treatment.

Total litter losses by resorption:

not specified

Early or late resorptions:

no effects observed

Description (incidence and severity):

The mean number of early resorptions were slightly high at 800 mg/kg/day and when compared with Controls the resultant post-implantation loss was high (p<0.05) and the live litter size was low (p<0.05). At 100 or 400 mg/kg/day the mean number of resorptions, the extent of the pre- and post- implantation loss and total number of live young were similar Controls and considered to be unaffected by treatment.

Dead fetuses:

no effects observed

Description (incidence and severity):

At 100 or 400 mg/kg/day the total number of live young were similar Controls and considered to be unaffected by treatment.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

effects observed, treatment-related

Description (incidence and severity):

At scheduled termination on Day 20 after mating, one female (no. 48) that received 400 mg/kg/day and one female (no. 79) that received 800 mg/kg/day were not pregnant. Macroscopic examination of female no. 48 revealed dark fluid in the uterine cervix, marked fluid distention of the uterus, and a blind portion of the vagina; female no. 79 showed no abnormality. All remaining animals were pregnant.

Details on maternal toxic effects:

Oral administration of ST 10 C 08 to pregnant Han Wistar rats during organogenesis and the fetal growth phase at 100, 400 and 800 mg/kg/day was generally well tolerated by maternal animals in life with effects on general condition limited to thin build for three animals at 800 mg/kg/day. There were no adverse effects on either maternal body weight gain, food consumption, thyroid hormones or macropathology.

Maternal thyroid weight however was significantly high at 400 or 800 mg/kg/day, and this correlated with the microscopic finding of follicular hypertrophy in some animals at these dose levels and circulating levels of Thyroid Stimulating hormone (TSH) were slightly high at 800 mg/kg/day. However, in the absence of an effect on thyroid weight or serum levels of either, Triiodothyronine (T3) or Thyroxine (T4), these findings were considered not to be adverse.
The gravid uterine weight at 800 mg/kg/day was significantly low when compared with Controls and this correlated with the high post-implantation loss, resultant low litter size and low fetal weight at this dose level; these parameters were unaffected at 100 or 400 mg/kg/day.

See the table of results in the attachments section.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

organ weights and organ / body weight ratios

pre and post implantation loss

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean placental weight was unaffected by treatment at dose levels up to and including 800 mg/kg/day. At 800 mg/kg/day the mean total litter weight was statistically significantly low when compared with Controls (p<0.01) and the mean overall fetal weight was also slightly low at 92% of Controls (p<0.05). The mean total litter weight and mean fetal weight were unaffected at 100 and 400 mg/kg/day.

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

The live litter size was low (p<0.05) at 800 mg/kg/day.
At 100 or 400 mg/kg/day the total number of live young were similar Controls and considered to be unaffected by treatment.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio was unaffected by treatment.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

At 800 mg/kg/day the mean total litter weight was statistically significantly low when compared with Controls (p<0.01) and the mean overall fetal weight was also slightly low at 92% of Controls (p<0.05). The mean total litter weight and mean fetal weight were unaffected at 100 and 400 mg/kg/day.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

The fetal ano-genital distance was unaffected by maternal treatment.

External malformations:

effects observed, treatment-related

Description (incidence and severity):

At 800 mg/kg/day the incidence of major abnormalities was high, including a number of skeletal and visceral abnormalities. In particular four fetuses from three litters exhibited Anophthalmia/Microphthalmia/Hydroceophaly, with the incidence for Hydrocephaly and Microphthalmia exceeding the historical control range.
At 400 mg/kg/day there were a number of unrelated major findings, at low incidence and the following were outside of the historical control data (HCD) range: absent tympanic annula, Agnathia, absent orbital socket(s), Microcephaly, Anury, imperforate anus.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

At 800 mg/kg/day the incidence of major abnormalities was high, including a number of skeletal and visceral abnormalities. In particular four fetuses from three litters exhibited Anophthalmia/Microphthalmia/Hydroceophaly, with the incidence for Hydrocephaly and Microphthalmia exceeding the historical control range.
At 400 mg/kg/day there were a number of unrelated major findings, at low incidence and the following were outside of the historical control data (HCD) range: absent tympanic annula, Agnathia, absent orbital socket(s), Microcephaly, Anury, imperforate anus.
At 400 and 800mg/kg/day there was a slight increase in incidence of shiny skin, 20 thoracolumbar vertebrae, incompletely ossified cranial centres and caudal vertebrae when compared with concurrent control and outside of HCD range (with the exception of cranial centres). These findings are indicative of fetal immaturity and are often associated with low mean fetal weight and are not considered adverse.
At 100 mg/kg/day one fetus in litter no. 34 had exencephaly which was within the historical control range, this fetus also had open eyelids which is considered secondary to the exencephaly. So, although the major abnormality of open eyelids exceeded HCD it is not considered to be treatment related as it is related to the exencephaly which was within HCD and was not observed at 400 or 800 mg/kg/day.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

At 800 mg/kg/day the incidence of major abnormalities was high, including a number of skeletal and visceral abnormalities. In particular four fetuses from three litters exhibited Anophthalmia/Microphthalmia/Hydroceophaly, with the incidence for Hydrocephaly and Microphthalmia exceeding the historical control range.
At 400 mg/kg/day there were a number of unrelated major findings, at low incidence and the following were outside of the historical control data (HCD) range: absent tympanic annula, Agnathia, absent orbital socket(s), Microcephaly, Anury, imperforate anus.

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

Detailed fetal examination revealed a low incidence of major predominately unrelated abnormalities at all dose levels the majority of which exceeded the historical control data range. The highest incidence of major abnormalities was seen at 800 mg/kg/day, in particular four fetuses from three litters exhibited Anophthalmia/Microphthalmia/Hydroceophaly. The increased post-implantation loss at 800 mg/kg/day and the high incidence of major abnormalities at this dose level are considered to be interrelated. At 400 mg/kg/day there were also a number of major findings, at low incidence and the following were outside of the historical control data (HCD) range: absent tympanic annula, Agnathia, absent orbital socket(s), Microcephaly, Anury, imperforate anus.
See the table of results in the attachments section.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

skeletal malformations

visceral malformations

Remarks on result:

other:

Fetal abnormalities

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

external: ear

external: eye

external: anus

skeletal: skull

visceral/soft tissue: central nervous system

visceral/soft tissue: eye

Description (incidence and severity):

At 800 mg/kg/day the incidence of major abnormalities was high, including a number of skeletal and visceral abnormalities: Four fetuses from three litters exhibited Anophthalmia/Microphthalmia/Hydroceophaly, the incidence for Hydrocephaly and Microphthalmia exceeding the historical control range. At 400 mg/kg/day there were also a number of unrelated major findings, at low incidence but a number were outside of the historical control data (HCD) range: absent tympanic annula, Agnathia, absent orbital socket(s), Microcephaly, Anury and imperforate anus.

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Formulation analysis:

The mean concentrations of formulations taken during the course of the study were within 4% of the nominal concentration, confirming the accuracy of formulation.
The difference from mean remained within 2%, confirming precise analysis, except for first Week where Group 2 had a difference from mean value of 6.70%. As the two individual values for Group 2 were within the +10/-15% acceptance criteria and the RME value was within the +10/-15% acceptance criteria, it is considered the formulations were accurately prepared. Procedural recoveries remained within the range established during the validation, confirming the continued accuracy of the analytical procedure.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, the maternal No-Observed-Adverse-Effect-Level (NOAEL) was 400 mg/kg bw/day (based on high post-implantation loss and low gravid uterine weight at 800 mg/kg/day) and the embryo-fetal No-Observed-Adverse-Effect-Level (NOAEL) was 400 mg/kg bw/day (based on major squeletal and visceral abnormalities at 800 mg/kg bw/day).

Executive summary:

In a prenatal developmental toxicity study performed according to OECD Guideline 414 and in compliance with GLP, three groups of 20 females received ST 10 C 08 at doses of 100, 400 or 800 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, corn oil at the same volume dose as treated groups. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.

Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating, blood samples were taken for thyroid hormone analysis and the gravid uterine weight and thyroid weight were recorded. Microscopic pathology investigations were also undertaken. Ano-genital distance was measured for fetuses and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

 

The mean concentrations of formulations taken during the course of the study were within 4% of the nominal concentration, confirming the accuracy of formulation.

 

Maternal responses

Administration of ST 10 C 08 at 100, 400 or 800 mg/kg/day resulted in high TSH levels at 800 mg/kg/day but no statistically significant effect on serum T3/T4 levels in pregnant female rats; TSH levels at 100 or 400 mg/kg/day were similar to Controls.

At routine physical examination the build for three females receiving 800 mg/kg/day was considered thin.
At 800 mg/kg/day mean absolute body weights from GD8-10 and on GD19/20 were statistically significantly low when compared with Controls; the overall body weight gain at 800 mg/kg/day was low at approximately 84% of Controls; however, this difference did not attain statistical significance. Body weight gain at 100 or 400 mg/kg/day was unaffected by treatment.

The gravid uterine weight was statistically significantly low when compared with Controls for females that received 800 mg/kg/day (p<0.01) but the maternal body weight gain, following adjustment for the gravid uterine weight, showed no adverse effect of treatment at dose levels up to and including 800 mg/kg/day.

At 800 mg/kg/day food consumption was low from GD6 to GD9; subsequent consumption was similar or slightly higher than Controls. At 400 mg/kg/day there were periods of slightly high consumption late gestation, but overall food consumption was unaffected by treatment.

Animals that received 400 or 800 mg/kg/day showed significantly high mean thyroid weight; a dose response was apparent.
No maternal macroscopic abnormalities detected at scheduled termination that were considered to relate to administration of ST 10 C 08.

Thyroid follicular cell hypertrophy (minimal severity) was seen in eight females administered 800 mg/kg/day and in one female at 400 mg/kg/day. This effect was concordant with effects occuring in a 90-day repeated dose toxicity study (Labcorp study No. 8449089, see reference to other study). In this study, minimal to slight follicular cell hypertrophy noted in males and females administered 400 or 800 mg/kg/day and males administered 100 mg/kg/day correlated with increased absolute and statistically significant body weight-adjusted thyroid gland weights for males. This finding is commonly encountered in rats in combination with centrilobular hypertrophy of the liver and is a consequence of hepatic microsomal enzyme induction causing increased clearance of thyroid hormones and feedback through the pituitary-thyroid axis (Curran and Degroot, 1991). Although microscopic examination of the liver was not evaluated in this study, we can assume that these thyroid follicular cell hypertrophy effects are a consequence of the induction of liver microsomal enzymes.

 

Litter responses

The mean number of early resorptions and post-implantation loss were high at 800 mg/kg/day and the resultant live litter size was low; litter data was unaffected by maternal treatment at 100 or 400 mg/kg/day.

At 800 mg/kg/day the mean total litter weight and the mean overall fetal weight were low; these parameters were unaffected at 100 or 400 mg/kg/day.

The fetal ano-genital distance was unaffected by maternal treatment at dose levels up to and including 800 mg/kg/day.

At 800 mg/kg/day the incidence of major abnormalities was high, including a number of skeletal and visceral abnormalities.  In particular four fetuses from three litters exhibited Anophthalmia/Microphthalmia/Hydroceophaly, the incidence for Hydrocephaly and Microphthalmia exceeding the historical control range. 

At 400 mg/kg/day there were also a number of unrelated major findings, at low incidence but a number were outside of the historical control data (HCD) range: absent tympanic annula, Agnathia, absent orbital socket(s), Microcephaly, Anury and imperforate anus.

At 400 and 800mg/kg/day there was a slight increase in incidence of shiny skin, 20 thoracolumbar vertebrae, incompletely ossified cranial centres and caudal vertebrae when compared with concurrent control and outside of HCD range (with the exception of cranial centres). These findings are indicative of fetal immaturity and are often associated with low mean fetal weight seen at these dose levels and are not considered adverse.

 

Based on the results of this study, the maternal No-Observed-Adverse-Effect-Level (NOAEL) was 400 mg/kg bw/day (based on high post-implantation loss and low gravid uterine weight at 800 mg/kg/day) and the embryo-fetal No-Observed-Adverse-Effect-Level (NOAEL) was 400 mg/kg bw/day (based on major squeletal and visceral abnormalities at 800 mg/kg bw/day).