Registration Dossier

Diss Factsheets

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-referenceopen allclose all
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 08 December 2020 to 11 August 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 408 without any relevant deviation.
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
Revised version 2018.
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
RccHan™:WIST
Details on species / strain selection:
RATIONALE FOR ANIMAL MODEL:
The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The RccHan™:WIST strain was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS Limited.
- Age at study initiation: 37-42 days
- Weight at study initiation: Males: 107-149 g; Females: 101-133 g
- Housing: Animals will be housed up to 4/cage in Polycarbonate body cages with a stainless steel mesh lid and wood based bedding changed at appropriate intervals.
- A plastic shelter and an aspen gnawing material was provided to each cage throughout the study and replaced when necessary.
- Diet: Teklad 2014C Diet, non-restricted (removed overnight before blood sampling for hematology or blood chemistry and during the period of urine collection)..
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals, non-restricted (except during urine collection).
- Acclimation period: 8 days before commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 40-70 %
- Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: 12 h dark / 12 h light
Route of administration:
oral: gavage
Details on route of administration:
RATIONALE FOR ROUTE OF ADMINISTRATION:
The oral gavage route of administration was chosen to simulate the conditions of possible human exposure during manufacture, handling and use of the test item.
Vehicle:
corn oil
Details on oral exposure:
VEHICLE
- Choice of vehicle: Corn oil
- Concentration in vehicle: 0, 20, 80 or 160 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg

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. Once the suspension was homogeneous it was magnetically stirred for a minimum of 20 minutes and throughout transfer of the suspension to the final containers
A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.
- Storage of formulation: Refrigerated (2 to 8°C).
- Frequency of preparation: Weekly
- Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined as part of another study, Labcorp 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).
- Before dosing: Formulations were mixed by 20-fold inversion and are stirred using a magnetic stirrer before dosing (for at least 20 minutes) and throughout the dosing procedure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ACHIEVED CONCENTRATION:
The samples taken from the Week 1 formulations were not analyzed because the laboratory could not be used for several weeks in December which coincided with when the samples should have been analyzed. When the issue was resolved the samples were out of stability. Additional samples were therefore taken from the formulations prepared for dosing in Week 6. Samples of each formulation prepared for administration in Weeks 6 and 12 of treatment were analyzed for achieved concentration of the test item.
Duration of treatment / exposure:
Minimum period: 13 weeks
Frequency of treatment:
Once daily at approximately the same time each day
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle (Group 1)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
400 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
800 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
10 animals per sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
RATIONALE FOR DOSE LEVEL SELECTION:
The doses used in this study (0, 100, 400 and 800 mg/kg/day) were selected in conjunction with the Sponsor. A combined repeated 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 was determined to be 800 and 100 mg/kg/day, respectively. The developmental NOAEL and NOEL was 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 = 1000 mg/g/day) would be expected to exhibit adverse effects that would not be consistent with animal welfare testing requirements. Therefore, the doses administered in this study were 0, 100, 400, and 800 mg/kg/day.

- Rationale for animal assignment (if not random): Randomly allocated on arrival.
- Fasting period before blood sampling for clinical biochemistry: Yes
Positive control:
Not applicable
Observations and examinations performed and frequency:
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 occupants. 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: Daily during the first week of treatment, twice weekly during Weeks 2 to 4 (middle and end of each week) and weekly thereafter at the following times in relation to dose administration:
• Pre-dose observation.
• At the end of dosing each group.
• One to two hours after completion of dosing of all groups.
• As late as possible in the working day
- Observations: Before treatment commenced and during each week of treatment, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day, by an observer unaware of the experimental group identities.
After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.
Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded one week before treatment commenced, on the day that treatment commenced (Week 0), weekly throughout the study and before necropsy.

FOOD CONSUMPTION: Yes
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the study.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Fluid intake was assessed by daily visual observation. No effect was observed and consequently quantitative measurements were not performed.

OPHTHALMOSCOPIC EXAMINATION: Yes
- The eyes of the animals were examined by means of a binocular indirect ophthalmoscope as follows:
• Pre-treatment: All animals of Groups 1, 2, 3 and 4 and spares.
• Week 12: All animals of Groups 1 and 4.
- Prior to each examination, the pupils of each animal were dilated using tropicamide ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Sampling was performed on the morning after overnight collection of urine. Animals were, therefore, also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures. Blood samples were collected at the following occasion:
• Occasion: Animals
• Week 13: All animals.
- Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL for haematology and 0.7 mL for blood chemistry) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined using a Bayer Advia 120 analyzer for haematology and into tubes containing lithium heparin anticoagulant and examined using a Roche Cobas 6000 Analyzer for blood chemistry.
- Parameters checked:
HAEMATOLOGY: Haematocrit (Hct), Haemoglobin concentration (Hb), Erythrocyte count (RBC), Absolute reticulocyte count (Retic), Mean cell haemoglobin (MCH), Mean cell volume (MCV), Mean cell haemoglobin concentration (MCHC), Red cell distribution width (RDW), Total leucocyte count (WBC), Platelet count (Plt) and Differential leucocyte count including neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M) and Large unstained cells (LUC). Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of Prothrombin time (PT) - using IL PT Fibrinogen reagent and Activated partial thromboplastin time (APTT) - using IL APTT reagent.

CLINICAL CHEMISTRY: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transferase (gGT), Urea, Total Bilirubin (Bili), Bile acid (Bi Ac), Blood urea nitrogen (BUN), Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), High-density lipoprotein (HDL), Low-density lipoprotein (LDL), Sodium (Na), Potassium (K), Total protein (Total Prot), Albumin (Alb), Triglycerides (Trig), Albumin/globulin ratio, Chloride (Cl), Calcium (Ca) and Inorganic phosphorus (Phos).

URINALYSIS: Yes
- Time schedule for collection of blood: Week 13: All main phase animals and at occasion for some animals.
- Conditions: overnight in an individual metabolism cage with deprivation of food and water
- Parameters checked: Clarity and Color (App) - by visual assessment, Volume (Vol) - using a measuring cylinder, pH - using a pH meter, Specific gravity (SG) - by direct refractometry using a SG meter, Bile pigments (Bili) and Blood pigments (UBld) using a multistix reagent strips and Protein - total (T-Prot) and concentration (Prot) and Glucose - total (T-Gluc) and concentration (U-Gluc) using a Roche Cobas 6000 analyzer.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Sensory reactivity and grip strength assessments were performed (before dosing) on all animals during Week 12 of treatment. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.
The following measurements, reflexes and responses were recorded: approach response, pinna reflex, auditory startle reflex, tail pinch response and grip strength.
- During Week 12 of treatment (before dosing), the motor activity of each animal was measured using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Covance. Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.

ENDOCRINE ENDPOINTS/THYROID HORMONE BIOSAMPLING: yes
The measurement of thyroxine (T4), triiodothyronine (T3), thyroid stimulating hormone (TSH), thyroid gland weight as well as levels of serum total cholesterol, low-density lipoproteins (LDL) and high-density lipoproteins (HDL) were assessed for effects on the thyroid.
Blood samples (1.0 mL) for thyroid hormone biosampling were collected at the following occasion from the sublingual vein:
• Occasion: Animals
• At necropsy: All animals.
- Conditions: No overnight deprivation of food.
- Anesthesic: Isoflurane. The animals did not recover from the anesthesia.
- Parameters checked: Triiodothyronine (T3), Thyroxine (T4) and Thyroid stimulating hormone (TSH).

ESTROUS CYCLES: Wet smears were taken from the vagina of all females (not decedents) using pipette lavage for four days before scheduled termination. The last smear was taken on the morning of necropsy. Smears were assessed to establish the stage of estrus (metestrus, diestrus, proestrus and estrus) at termination and were used to assist in the histological evaluation of estrogen sensitive tissues.
Sacrifice and pathology:
SACRIFICE: Animals surviving until the end of the scheduled study period were killed following 13 weeks of treatment. Animals were killed by carbon dioxide asphyxiation with subsequent exsanguination.

GROSS PATHOLOGY: Yes; All 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 appropriate fixative.

ORGAN WEIGHTS: For bilateral organs, left and right organs were weighed together, unless specified in Table 7.5.1/1. Requisite organs were weighed for animals killed at scheduled intervals.

HISTOPATHOLOGY: Yes
- Fixation: Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of testes which are preserved in modified Davidson’s fluid and eyes which are preserved in Davidson’s fluid.
- Histology:
Processing: 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.
• Full List: All animals killed or dying prematurely and animals of Groups 1 and 4 killed at a scheduled interval.
• Liver, kidneys, thyroids and abnormalities: All main study animals of Groups 2 and 3 killed at a scheduled interval.
Routine staining: Sections were stained with hematoxylin and eosin.
Special staining: Additional 4-5 µm sections were prepared from the kidneys of all males from all groups and stained by immunohistochemistry for alpha-2u-globulin.

- Light microscopy: Tissues preserved for examination were examined as specified in Table 7.5.1/1 for:
• Premature deaths: All animals from all groups.
• Scheduled kill: All animals of Groups 1 and 4.
• Scheduled kill: Liver, kidneys, thyroid and abnormalities were examined for all animals of Groups 2 and 3.
- Detailed qualitative examination were made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage specificity of testicular findings were noted.
Optional endpoint(s):
SPERM ANALYSIS: yes
A sperm sample was taken from the left vas deferens at necropsy of all males killed after 13 weeks of treatment and assessed for motility using a computer assisted sperm analyzer (CASA). These data are not reported. The residual sperm samples taken from the left vas deferens were retained in neutral buffered formalin. The left testis and left cauda epididymis were also weighed and retained deep frozen ( 10 to 30°C).
> As no treatment-related changes were identified from organ weight or histopathology investigations, an assessment of sperm count and sperm morphology was not performed and all retained samples were discarded when the report was finalized. The results of the sperm motility assessment were retained in the raw data but not reported.
Statistics:
See "Any other information on materials and methods incl. tables"
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Increased salivation, chin rubbing and paddling of forepaws after dosing were seen in animals receiving 800 mg/kg bw/day and, to a lesser extent in animals receiving 400 mg/kg bw/day (at this dose level paddling of paws was only observed in females). These signs are often seen in animals dosed via the oral gavage route and are considered to relate to palatability of formulations rather than a direct effect of the test item.
The type, incidence and distribution of other clinical signs observed for either sex during the study did not indicate any obvious association with treatment. This included observations made during the assessment of the animals for behavior during handling and after being placed in a standard arena.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no unscheduled deaths during the study due to treatment with ST 10 C 08.
Female No. 115 receiving 100 mg/kg/day was euthanised for welfare reasons on Day 6 of treatment, after biting off and swallowing a portion of the canula during dosing. The animal had shown no clinical or post dose signs prior to death. Macroscopic examination at necropsy confirmed that the portion of canula was lodged in the animal’s lower oesophagus/stomach and it was also noted that several small (
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No statistically significant differences in body weights for males or females were observed between any treatment group and the control group on a weekly basis.
Overall body weight gain was slightly low for males receiving 800 mg/kg bw/day, at 89% of Controls (not statistically significant). Similarly, overall body weight gains were lower for females that received 400 or 800 mg/kg bw/day, at 89% or 87% of Controls, respectively (p<0.05).
Body weight at 100 mg/kg bw/day was unaffected by treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was unaffected by administration of ST 10 C 08 at dose levels up to and including 800 mg/kg bw/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
No effect was observed and consequently quantitative measurements were not performed.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmic examination of animals receiving 800 mg/kg bw/day revealed no treatment related effects.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Haematological examination revealed low haematocrit, haemoglobin and reticulocyte counts for females at 400 or 800 mg/kg bw/day. There was no corresponding effect on erythrocyte count at either of these dose levels in females and erythrocyte parameters for treated males were similar to Controls.
High lymphocyte counts were apparent for males and females that received 800 mg/kg bw/day and for females at 400 mg/kg bw/day, with basophil counts high in males at 400 or 800 mg/kg bw/day and large unstained cell counts high in females at 800 mg/kg bw/day. These changes resulted in a concomitant increase in total white blood cell counts in females that received 400 mg/kg bw/day and in males and females at 800 mg/kg bw/day.
Lower activated partial thromboplastin times (APTT), compared with control, was apparent for males and females at 400 or 800 mg/kg bw/day.
While it cannot be clearly established that any of these individual changes represent an adverse effect, collectively, they indicate evidence that exposure to the test substance at the doses of 400 and 800 mg/kg bw/day are not well tolerated. At 100 mg/kg bw/day there was no evidence of treatment-related hematological for either males or females. A statistically significant decrease in AST and ALT was observed for males however, as these values were decreased rather than increased this is not considered an adverse effect.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Biochemical examination of the blood plasma revealed high gamma glutamyl transferase activities in males that received 400 mg/kg bw/day and in males and females at 800 mg/kg bw/day. Alkaline phosphatase and alanine aminotransferase activities were low in males at all dose levels with individual values as low as <5 U/L for two animals treated at 800 mg/kg bw/day. Reductions in enzyme activities are usually considered of no toxicological importance but the very low values reported for some animals at 800 mg/kg bw/day are unusual and may be an indication of metabolic disturbance. Aspartate amino transferase activities were low in females that received 800 mg/kg bw/day but given that no other enzymes were affected in the females, this finding appears incidental and unrelated to treatment. An increase in triglyceride concentrations was also seen in females receiving 400 or 800 mg/kg bw/day. Males at 800 mg/kg bw/day had high plasma urea concentrations.

Plasma calcium concentrations were high in males and females receiving 400 or 800 mg/kg bw/day as were phosphorus concentrations in males at these dose level. Plasma chloride concentrations were low in males receiving 400 mg/kg bw/day and males and females receiving 800 mg/kg bw/day.

Effects were apparent on plasma proteins with total protein concentrations increased in males and females receiving 400 or 800 mg/kg bw/day, which despite higher albumin concentrations in both sexes receiving 800 mg/kg bw/day, resulted in decreases in albumin to globulin ratios in both sexes receiving 400 or 800 mg/kg bw/day.

When compared with the controls, statistically significant differences were seen in the concentrations of bile acids in the plasma. In males the concentrations were low at 400 or 800 mg/kg bw/day and in females the concentrations were high in treated groups but with no dose response. The individual results were very variable, particularly in the females (Animal No.120 at 100 mg/kg bw/day: 101 µmol/L and Animal No.135 at 400 mg/kg bw/day: 158 µmol/L; both values being higher than any reported for females at 800 mg/kg bw/day). Consequently, it was considered that these changes were unlikely to have arisen as a result of treatment with ST 10 C 08 but were due to biological variation.
There were no changes in plasma biochemistry for animals receiving 100 mg/kg bw/day.
While it cannot be clearly established that any of these individual changes represent an adverse effect, collectively, they indicate evidence that exposure to the test substance at the doses of 400 and 800 mg/kg/day are not well tolerated. There were no other clinical chemistry differences between males at 100 mg/kg bw/day and the concurrent Control group. In females at 100 mg/kg bw/day there was an increase in bile acids that was statistically significant when compared with Controls but was clearly not dose-dependent. Additionally, a statistically significant increase in LDL cholesterol was observed for females from at 100 mg/kg bw/day however, no changes in LDL cholesterol were observed in males from the low or middle dose group. Finally, a statistically significant decrease in A/G ratio was observed in low dose females but not in low dose males.
Endocrine findings:
effects observed, treatment-related
Description (incidence and severity):
There was no effect of treatment on estrous cycles as assessed during the four days prior to necropsy, with all females showing estrus before termination.
Males and females at 400 or 800 mg/kg bw/day had high cholesterol and high-density lipoprotein concentrations, with low-density lipoprotein concentrations also high in males at 800 mg/kg bw/day and in all groups of ST 10 C 08 treated females.
In the thyroids, minimal to slight follicular cell hypertrophy was noted in males and females administered 400 or 800 mg/kg bw/day and males administered 100 mg/kg bw/day, with the higher incidence and severity occurring in males. This 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). Since the rodent thyroid gland is many times more sensitive to disruptions of the hormonal control of the thyroid gland than that of man, this mechanism of action is rodent specific and has little toxicological relevance to man; consequently, this change is considered to be non-adverse.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
With the exception of volume, all urinary parameters in treated males were either decreased (pH) or increased (specific gravity, total protein, protein, total glucose and urinary glucose) to values that were statistically different from the values observed in the concurrent control group. Most impressive were the increases in total protein (mg) to values that were +90%, +213%, and +279% of Controls in the low, middle and high dose groups, respectively.
Similarly, the protein concentration (g/L) was increased at +118%, +250%, and +234% of Controls, in the low, middle, and high dose groups, respectively. In females, an increase in total protein (mg) was also observed but was attributed to an increased production of urine as the concentration (g/L) of protein was not increased in any of the treatment groups. This effect on urinary protein levels in treated males is consistent with the kidney pathology for this sex.
The appearance of the urine tended to be darker in the ST 10 C 08 treated groups of males, the urine of three male animals was described as orange in colour.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Sensory reactivity and grip strength were unaffected by treatment.
There was no effect of treatment on motor activity for either sex.
Males receiving 400 or 800 mg/kg bw/day were initially more active than the Controls with statistical significance attained for both rearing activity (high beam) and ambulatory (low beam); rearing activity was also statistically significantly high for males at 100 mg/kg bw/day during this period. However, mean values for high beam breaks showed no consistent dose relationship and both high and low beam scores of the treated males were within the historical control data range (HCD). Subsequent mean scores for motor activity tended to be slightly lower than Controls as the assessment period progressed, with statistical significance (p<0.05) achieved at the 18-minute interval (high beam at all dose levels), the 30-minute interval (high beam at 800 mg/kg bw/day) the 48-minute interval (high and low beam at 800 mg/kg bw/day) and the 54-minute interval (low beam at 800 mg/kg bw/day), although all these scores were within the HCD range. Intergroup differences for mean overall activity scores for the one-hour testing period were small and values for treated animals did not attain statistical significance when compared with Control values. Overall, the pattern of behavior seen in this assessment was as expected, with high exploratory activity being observed initially followed by lower exploratory activity as the animals became familiar with their new environment. The occasional intergroup differences observed during this assessment were attributed to natural variation and were considered unrelated to treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
After 13 weeks of treatment, statistically significant adjusted mean organ weight increases were apparent for the liver, thyroids, kidneys, adrenals and ovaries:
- The body weight-adjusted liver weights of males and females administered 100, 400 or 800 mg/kg bw/day were statistically significantly higher than Controls; a dose response was apparent. The absolute liver weights were also high when compared with Controls.
- The body weight-adjusted thyroid weights of males administered 400 or 800 mg/kg bw/day were statistically significantly high when compared with Controls; absolute weights were also high, and a dose response was apparent. The increase in the thyroid weights was also present in females administered 400 or 800 mg/kg bw/day also showed slightly high thyroid weight when compared with Controls but the difference did not attain statistical significance.
- The body weight-adjusted kidney weights of males at 100, 400 or 800 mg/kg bw/day were statistically significantly high when compared with Controls (p<0.01); a dose response was apparent.
- The body weight-adjusted adrenal weights of males administered 400 or 800 mg/kg bw/day were statistically significantly high (p<0.01), with the extent of the increase being dose related.
- The body weight-adjusted ovary weights of females administered 400 or 800 mg/kg bw/day were statistically significantly higher than in controls. This did not correlate with any macroscopic or microscopic changes and was considered incidental.

After 13 weeks of treatment, statistically significant adjusted mean organ weight decreases were noted for the epididymides and heart, without any macroscopic or microscopic correlations attributable to treatment of males that received 800 mg/kg bw/day; this difference in heart weight was not apparent in females at this dose level.

See Table 7.5.1/2 in the "Any other information on results incl. tables" section.
All other differences in organ weight parameters were consistent with normal variation and considered incidental. These differences were characterized by one or more of the following: inconsistency between sexes; lack of a dose relationship or correlative findings; and/or the magnitude was considered small.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
After 13 weeks of treatment, test item-related macroscopic findings were present in the liver and kidneys. Abnormally dark coloration of the liver and kidneys was present in females administered 400 or 800 mg/kg bw/day. See Table 7.5.1/3 in the "Any other information on results incl. tables" section. All other macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or were as expected for animals of this age and strain. Therefore, they were considered not test item related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
After 13 weeks of treatment, test item related microscopic findings were present in the liver, thyroids and kidneys:
- In the liver, minimal to slight centrilobular hypertrophy was present in males and females administered 800 mg/kg bw/day and in females administered 100 or 400 mg/kg bw/day, with the higher incidence and severity occurring in females. Considering the correlation between the increased absolute and statistically significant body weight-adjusted liver weights, the macroscopic and the biochemistry changes, these changes are most likely an indication of liver metabolic alterations.
- In the thyroids, minimal to slight follicular cell hypertrophy was noted in males and females administered 400 or 800 mg/kg bw/day and males administered 100 mg/kg bw/day, with the higher incidence and severity occurring in males. This 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). Since the rodent thyroid gland is many times more sensitive to disruptions of the hormonal control of the thyroid gland than that of man, this mechanism of action is rodent specific and has little toxicological relevance to man; consequently, this change is considered to be non-adverse.
- In the kidneys, slight to moderate accumulation of hyaline droplets together with minimal to moderate tubular basophilia and granular casts were observed in males administered 100, 400 or 800 mg/kg bw/day. This correlated with the increased absolute and statistically significant body weight-adjusted kidney weights. Additional kidney sections subject to immunohistochemistry confirmed the nature of droplets to be alpha-2u-globulin. Alpha 2u-globulin nephropathy syndrome is considered to be male rat specific (Swenberg 1993) and adverse but not relevant to man. This pathology correlated with the increased urinary protein in males which was not evident in females.
Minimal to slight tubular pigment was present in females administered 400 or 800 mg/kg bw/day, this correlated with the abnormally dark bilateral kidney coloration at macroscopic examination. Kidney sections stained positive for Schmorl’s ferricyanide although this may indicate lipofuscin, the specificity of the stain is not sufficient to indicate increased “wear and tear” of the affected cells as it can also stain melanin, bile, haematoidin, argentaffin cells, chromaffin, and sulphydryl groups. There is also the possibility that this pigmentation could represent accumulation of the test item or metabolite(s) and we are unable to predict how this would interact with the stain. Clinically females showed increased urinary output resulting in high total urinary protein and glucose, however protein and glucose urinary concentrations were similar to Controls. In the absence of any degenerative changes in the female kidneys, changes in female kidney weight or any blood chemistry changes in females that could indicate kidney damage the tubular pigmentation in the female animals is considered to be non-adverse.
See Table 7.5.1/4 in the "Any other information on results incl. tables" section.

All other microscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or their severity was as expected for this animal age and strain. Therefore, they were considered not test article related.
The testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.
Macroscopically at necropsy two small firm and pale masses were noted on the non-glandular mucosa of the stomach in two females given 800 mg/kg bw/day (Animals No. 23 and No.128). The masses were correlated with a squamous cyst which is most likely to be of congenital origin in Animal No. 128 and focal slight hyperplasia of the nonglandular stomach in animal No. 123. On microscopic examination, minimal diffuse hyperplasia of the non-glandular stomach was also noted in one female administered 800 mg/kg bw/day, without any macroscopic correlations. Hyperplasia of the nonglandular stomach in two females administered 800 mg/kg bw/day is most likely incidental, given its absence in males and its low incidence.
Two special stains were applied to kidneys from control and treated representative females for further evaluation of the tubular pigment. Schmorls Ferricyanide special staining was positive for lipofuscin, staining with Perls Prussian Blue was negative for ferric iron in all representative females; this correlated with the abnormally dark bilateral kidney coloration and clinically observed increase in total urinary protein present across all treated females.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
Formulation analysis: Following re-injections and contingency analysis for Week 6 Groups 1 to 3, the mean concentrations for both Week 6 and Week 12 were within 5% of the nominal concentration, confirming the accuracy of formulation. The difference from mean and coefficient of variations remained within 3%, confirming precise analysis. Procedural recoveries remained within the range established during the validation, confirming the continued accuracy of the analytical procedure.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Key result
Critical effects observed:
no

Table 7.5.1/2. Effects in Organ Weight Parameters – Terminal Sacrifice After 13 Weeks of Treatment



































































































































































































































































Test item

ST 10 C 08



Sex



Males



Females



Dose Level (mg/kg/day)



0



100



400



800



0



100



400



800



Adrenals



 



 



 



 



 



 



 



 



Absolute Weight (g)



0.053



104



113



113



0.062



103



110



100



Body Weight Adjusted (%)



0.052



106



115**



117**



0.060



105



115



107



Epididymides



 



 



 



 



 



 



 



 



Absolute Weight (g)



1.396



95



93



88



NA



NA



NA



NA



Body Weight Adjusted (%)



1.397



95



93



88**



NA



NA



NA



NA



Heart



 



 



 



 



 



 



 



 



Absolute Weight (g)



1.091



94



92



83



0.754



96



92



94



Body Weight Adjusted (%)



1.040



100



97



91*



0.736



97



96



99



Kidneys



 



 



 



 



 



 



 



 



Absolute Weight (g)



2.092



103



116



122



1.451



98



95



96



Body Weight Adjusted (%)



1.942



113**



126**



137**



1.396



100



102



103



Liver



 



 



 



 



 



 



 



 



Absolute Weight (g)



12.701



106



134



150



8.038



107



140



196



Body Weight Adjusted (%)



11.549



119**



148**



172**



7.741



109**



149**



207**



Thyroids



 



 



 



 



 



 



 



 



Absolute Weight (g)



0.017



100



135



141



0.017



100



112



112



Body Weight Adjusted (%)



0.016



106



144**



150**



0.017



100



112



118



Ovaries



 



 



 



 



 



 



 



 



Absolute Weight (g)



NA



NA



NA



NA



0.092



108



114



125



Body Weight Adjusted (%)



NA



NA



NA



NA



0.087



110



124*



137**



NA = Not applicable.


* = Statistically significant difference (absolute or relative) compared with respective control mean value.


Note: Values for absolute weight and ratio of organ weights (relative to body weight) for dosed groups expressed as percentage control mean value.



 


Table 7.5.1/3. Macroscopic Findings – Terminal Sacrifice After 13 Weeks of Treatment



























































































Test item

ST 10 C 08



Sex



Males



Females



Dose Level (mg/kg/day)



0



100



400



800



0



100



400



800



Kidneys



 



 



 



 



 



 



 



 



Number Examined



10



10



10



10



10



9



10



10



  Abnormal Color (dark)



0



0



0



0



0



0



2



3



Liver



 



 



 



 



 



 



 



 



Number Examined



10



10



10



10



10



9



10



10



  Abnormal Color (dark)



0



0



0



0



0



0



6



9



 


Table 7.5.1/4. Microscopic Findings – Terminal Sacrifice After 13 Weeks of Treatment























































































































































































































































































































Test item

ST 10 C 08



Sex



Males



Females



Dose Level (mg/kg/day)



0



100



400



800



0



100



400



800



Liver



 



 



 



 



 



 



 



 



Number Examined



10



10



10



10



10



10



10



10



  Centrilobular Hypertrophy



 



 



 



 



 



 



 



 



Minimal



0



0



0



3



0



2



7



6



Slight



0



0



0



1



0



0



0



3



Thyroids



 



 



 



 



 



 



 



 



Number Examined



10



10



10



10



10



10



10



10



  Hypertrophy, Follicular Cells



 



 



 



 



 



 



 



 



Minimal



0



3



4



0



0



0



2



5



Slight



0



0



2



10



0



0



0



0



Kidney



 



 



 



 



 



 



 



 



Number Examined



10



10



10



10



10



10



10



10



  Accumulation, Hyaline Droplets



 



 



 



 



 



 



 



 



Slight



0



10



2



1



0



0



0



0



Moderate



0



0



8



9



0



0



0



0



  Basophilia, Tubular



 



 



 



 



 



 



 



 



Minimal



0



7



5



7



0



0



0



0



Slight



0



0



4



1



0



0



0



0



Moderate



0



1



1



1



0



0



0



0



  Cast, Granular



 



 



 



 



 



 



 



 



Minimal



0



4



2



3



0



0



0



0



Slight



0



0



2



0



0



0



0



0



Moderate



0



0



0



1



0



0



0



0



  Pigment, Tubular



 



 



 



 



 



 



 



 



Minimal



0



0



0



0



0



0



7



6



Slight



0



0



0



0



0



0



0



3


Conclusions:
Administration of ST 10 C 08 via oral gavage to Han Wistar rats at 100, 400 or 800 mg/kg bw/day had no adverse effect on clinical condition, sensory reactivity, grip strength, motor activity, food consumption or estrous cycles and there were no test item effects observed during the ophthalmic examination.

After 13 weeks of treatment, non-adverse test item related microscopic findings were present in the liver (centrilobular hypertrophy), thyroid glands (follicular cell hypertrophy) and kidneys (tubular pigment). Adverse findings were present in the kidneys of all treated males (accumulation of hyaline droplets, tubular basophilia and granular casts), with a corresponding increase in urinary protein, however this is an alpha 2u-globulin nephropathy syndrome which is considered to be a rat specific toxicity which is not relevant to man. However, increased body weight adjusted liver weights were also apparent for both sexes within this study at dose level of 100 mg/kg bw/day and above. Whilst these increases in liver weights were not associated with any adverse histopathological change, all histopathological liver findings were considered to be adaptive in nature, the magnitude of these increases for liver weights at 400 or 800 mg/kg bw/day were sufficient for this finding to be regarded as an adverse effect at these dose levels.
It was therefore concluded a dose level of 100 mg/kg bw/day represented the No-Observed-Adverse-Effect-Level (NOAEL).
Executive summary:

In a repeated dose toxicity study performed in accordance with OECD test guideline No. 408 and in compliance with GLP,  three groups, each comprising ten male and ten female Han Wistar (RccHan™;WIST) rats received ST 10 C 08 at doses of 100, 400 or 800 mg/kg bw/day by oral gavage administration. A similarly constituted control group received the vehicle, corn oil, over the same treatment period and at the same volume dose as the treated groups.


During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, visual water consumption, ophthalmic examination, hematology (peripheral blood), blood chemistry, stage of estrous cycle, organ weight, macropathology and histopathology investigations were undertaken.


 


 


Sensory reactivity, grip strength, motor activity, food consumption and estrus cycles were unaffected by treatment at any dose level and there were no test item effects observed during the ophthalmic examamination. Salivation, chin rubbing and paddling of forepaws were seen in animals receiving 800 mg/kg bw/day and, to a lesser extent, in animals receiving 400 mg/kg bw/day.


 


No statistically significant differences in body weights for males or females were observed between any treatment group and the control group on a weekly basis, however, overall body weight gain was slightly low for males at 800 mg/kg bw/day, at 89% of Controls (not statistically significant). Similarly, overall body weight gains were lower for females that received 400 or 800 mg/kg bw/day, at 89% or 87% of Controls, respectively (p<0.05). Food consumption was not affected in any treatment group.


 


Haematological examination showed a dose-dependent reduction in haematocrit, haemoglobin and reticulocyte counts for females that was statistically significant at 400 and 800 mg/kg bw/day. These variables were not affected in males at any treatment level. White blood cells and lymphocytes counts were high at 400 and 800 mg/kg bw/day, with statistically significance attained for males at 800 mg/kg bw/day (p<0.01), females at 400 mg/kg bw/day (p<0.05), and females at 800 mg/kg bw/day (p<0.01). Basophil counts were increased at 400 and 800 mg/kg bw/day for males (p<0.05) but not in treated females. Large unstained cell counts were increased in females at 800 mg/kg bw/day (p<0.05). Reduced activated partial thromboplastin time (APTT) was apparent for both males and females at 400 mg/kg bw/day (p<0.05) and 800 mg/kg bw/day (p<0.01).


A statistically significant decrease in alkaline phosphatase and alanine aminotransferase was observed in males from all treatment groups but no changes were observed in these variables from females in any treatment group.


A statistically significant (p<0.05) decrease in aspartate aminotransferase was observed in females at 800 mg/kg bw/day.


 


Biochemical examination of the blood plasma indicated high gamma glutamyl transferase activity in males that received 400 or 800 mg/kg bw/day and in females at 800 mg/kg bw/day (all p<0.01).


Total bilirubin concentration different from controls at 400 and 800 mg/kg bw/day for males and all treated females (p<0.05) however, this difference was statistical in nature as the mean values were identical in all treatment groups.


A dose-dependent decrease was observed in bile acid concentrations of males that was statistically significant at 400 mg/kg bw/day (p<0.05) and at 800 mg/kg bw/day (p<0.01).  In contrast, increased bile acids were observed for females at 100 and 400 mg/kg bw/day, but these differences did not attain statistical significance. 


Urea and blood urea nitrogen were elevated in males at 800 mg/kg bw/day (p<0.05), but these changes were not in treated females.


A dose-dependent increase in total cholesterol concentration was observed in treated males with statistically significance attained at 400 mg/kg bw/day (p<0.05) and 800 mg/kg bw/day (p<0.01).  Similarly, a dose-dependent increase in total cholesterol concentration was observed in all females that was statistically significant at 400 and 800 mg/kg bw/day (p<0.01). A dose-dependent increase in HDL cholesterol was observed in all male and female treatment groups that was that was statistically significant at 400 and 800 mg/kg bw/day (p<0.01). LDL cholesterol was significantly elevated in males at 800 mg/kg bw/day (p<0.01).  In females, LDL cholesterol was increased in a dose-dependent manner that was statistically significant at 100 mg/kg bw/day (p<0.05) and at 400 and 800 mg/kg bw/day (p<0.01).


Plasma triglycerides were increased in females in a dose-dependent manner and were statistically significant at 400 and 800 mg/kg bw/day (p<0.01); no differences were observed in male treatment groups.


Plasma concentrations of chloride were decreased in all male treatment groups, but the decrease was only statistically significant at 400 and 800 mg/kg bw/day (p<0.05). Chloride concentrations were also significantly low for females at 800 mg/kg bw/day group (p<0.01). A modest but statistically significant increase in plasma calcium concentrations was observed in males and females at 400 and 800 mg/kg bw/day (p<0.01). A statistically significant increase in plasma phosphate concentrations was observed in males at 400 and 800 mg/kg bw/day (p<0.05). No changes were observed in serum phosphate concentrations for treated females. Plasma total protein concentrations were significantly elevated at 400 mg/kg bw/day (males p<0.05; females p<0.01) and 800 mg/kg bw/day (males and females p<0.01); the magnitude was greater for females. Albumin concentrations were also significantly increased in males and females at 800 mg/kg bw/day (p<0.05 and p<0.01, respectively). Correspondingly, the A/G ratio was low in females at 100 mg/kg bw/day (p < 0.05) and in males and females at 400 and 800 mg/kg bw/day (p<0.01).


 


With the exception of volume, all urinary parameters in treated males were either decreased (pH) or increased (specific gravity, total protein, protein, total glucose and urinary glucose) to values that were statistically different from the values observed in the concurrent control group. Most impressive were the increases in total protein (mg) to values that were +90%, +213%, and +279% of Controls in the low, middle and high dose groups, respectively. Similarly, the protein concentration (g/L) was increased at +118%, +250%, and +234% of Controls, in the low, middle, and high dose groups, respectively. In females, an increase in total protein (mg) was also observed but was attributed to an increased production of urine as the concentration (g/L) of protein was not increased in any of the treatment groups. This effect on urinary protein levels in treated males is consistent with the kidney pathology for this sex.


 


The body weight-adjusted liver weights of males and females administered 100, 400 or 800 mg/kg bw/day were significantly high when compared with Controls in a dose related manner. Macroscopic examination revealed abnormally dark coloration of the liver for females that received 400 or 800 mg/kg bw/day and histopathological examination revealed minimal to slight centrilobular hypertrophy in males and females at 800 mg/kg bw/day and in females at 100 or 400 mg/kg bw/day, with the higher incidence and severity occurring in females.


 


The body weight-adjusted thyroid weights of males administered 400 or 800 mg/kg bw/day were significantly high (p<0.01) when compared with Controls in a dose related manner. Body weight-adjusted thyroid weight was not affected in females from any treatment groups. Histopathological examination revealed minimal to slight follicular cell hypertrophy in males and females at 400 or 800 mg/kg bw/day and males at 100 mg/kg bw/day, with the higher incidence and severity occurring in males.


 


When compared with Controls there was a dose dependent increase in body weight-adjusted kidney weights for males at all dose levels (p<0.01) but no changes in any of the treated females. Macroscopic examination after 13 weeks of treatment also revealed abnormally dark coloration of the kidneys for females that received 400 or 800 mg/kg bw/day. Histopathological examination revealed slight to moderate accumulation of hyaline droplets together with minimal to moderate tubular basophilia and granular casts for males administered 100, 400 or 800 mg/kg bw/day. Additional kidney sections stained by immunohistochemistry confirmed the nature of droplets to be alpha-2u-globulin; this correlated with the increased urinary protein recorded for treated males. Minimal to slight tubular pigment was present in females administered 400 or 800 mg/kg bw/day; the pigment was confirmed to be lipofuscin by Schmorls Ferricyanide staining.


 


A dose-dependent increase in body weight-adjusted adrenal weights was observed in males at 400 and 800 mg/kg bw/day (p<0.01) but no changes were observed in any of the female treatment groups. Histopathological examination of the adrenals did not present any notable findings.


 


Body weight-adjusted heart weight was decreased in males at 800 mg/kg bw/day (p<0.05), but no changes were observed in any of the female treatment groups. Histopathological examination of the hearts did not reveal any noteworthy findings.


 


A dose-dependent decrease was observed in the body weight-adjusted epididymal weight that with statistical significance attained at 800 mg/kg bw/day (p<0.01). No noteworthy observations were present when examined microscopically. 


 


A dose-dependent increase in body weight-adjusted ovary weights was statistically significant at 400 mg/kg bw/day (p<0.05) and 800 mg/kg bw/day (p<0.01) treatment groups. No noteworthy observations were present when examined microscopically. 


 


 


Administration of ST 10 C 08 via oral gavage to Han Wistar rats at 100, 400 or 800 mg/kg bw/day had no adverse effect on clinical condition, sensory reactivity, grip strength, motor activity, food consumption or estrous cycles and there were no test item effects observed during the ophthalmic examination.


After 13 weeks of treatment, non-adverse test item related microscopic findings were present in the liver (centrilobular hypertrophy), thyroid glands (follicular cell hypertrophy) and kidneys (tubular pigment). Adverse findings were present in the kidneys of all treated males (accumulation of hyaline droplets, tubular basophilia and granular casts), with a corresponding increase in urinary protein, however this is an alpha 2u-globulin nephropathy syndrome which is considered to be a rat specific toxicity which is not relevant to man. However, increased body weight adjusted liver weights were also apparent for both sexes within this study at dose level of 100 mg/kg bw/day and above. Whilst these increases in liver weights were not associated with any adverse histopathological change, all histopathological liver findings were considered to be adaptive in nature, the magnitude of these increases for liver weights at 400 or 800 mg/kg bw/day were sufficient for this finding to be regarded as an adverse effect at these dose levels.


It was therefore concluded a dose level of 100 mg/kg bw/day represented the No-Observed-Adverse-Effect-Level (NOAEL).

Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2008-09-29 to 2009-08-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 422 and in compliance with GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
OECD GLP (inspected on 05th to 09th and 26th to 30th November 2007, Signed on 12th November 2008)
Limit test:
no
Species:
rat
Strain:
other: HanRcc: WIST(SPF)
Details on species / strain selection:
Recognized by international guidelines as a recommended test system.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories Ltd. Laboratory Animal Services Wölferstrasse 44414 Füllinsdorf / Switzerland
- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 284 to 318 g; Females: 178 to 214 g
- Fasting period before study: none
- Housing: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ Schill AG, 4132 Muttenz / Switzerland). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular oestrus cycles.
- Diet (e.g. ad libitum): Pelleted standard Kliba Nafag 3433 rat/mouse maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum (batch no. 31/08).
- Water (e.g. ad libitum): Community tap-water from Füllinsdorf was available ad libitum in water bottles
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): Air-conditioned with 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light / 12-hour dark cycle with music during the light period

IN-LIFE DATES: From: 2008-09-28 To: 2009-08-27
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was weighed into a glass beaker on a tarred precision balance and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added.
Separate formulations were prepared for each concentration. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): 24897436
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first treatment day, one sample (middle) from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (7 days). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose
formulations were frozen (-20 ± 5 °C) and delivered on dry ice to Dr. D. Flade (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis. The samples were analysed by GC coupled to an FI detector following an analytical procedure provided by the Sponsor and adapted at Harlan Laboratories. The test item was used as the analytical standard. Analysed samples were not discarded without written consent from the study director.

The identity of the test material was confirmed by its retention time, which was similar to that measured in the working standards. The application formulations investigated during the study were found to comprise the test material in the range of 91.7% to 108.9% and thus, the required content limit of ±20% with reference to the nominal concentration was met. The homogeneous distribution of the test material in the preparations was approved because single results did not
deviate more than 5.9% (<15%) from the corresponding mean. The application formulations were considered to be stable for at least 7 days when kept at room temperature.
Duration of treatment / exposure:
Males: minimum 4 weeks. Females: approximately 7 weeks.
Frequency of treatment:
Daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Dose / conc.:
800 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a previous dose range finding toxicity study in Han Wistar Rats, Harlan Laboratories Study C05595, using dose levels of 100, 300, and 1000 mg/kg bw/day.
- Rationale for animal assignment (if not random): Computer-generated random algorithm. In addition body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
Positive control:
none
Observations and examinations performed and frequency:
VIABILITY / MORTALITYS: Yes
- Time schedule: Twice daily

CLINICAL SIGNS: Yes
- Time schedule: Daily cage-side clinical observations (once daily during acclimatization and up to day of necropsy).
Additionally females were observed for signs of difficult or prolonged parturition, and behavioural abnormalities in nesting and nursing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to the first administration of the test item and weekly thereafter, performed outside the home cage. Animals were observed
- Parameters examined: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behaviour were also reported.

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded daily from treatment start to day of necropsy.

FOOD CONSUMPTION:
Males: Weekly during pre-pairing and after pairing periods
Females: Pre-pairing period days 1-8, 8-14 and 14-16; gestation days 0-7, 7-14 and 14-21 post coitum, and days 1-4 post partum.
No food consumption was recorded during the pairing period.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were obtained on the day before or on the day of the scheduled necropsy from
5 males from each group. Blood samples from 5 lactating females from each group were obtained on day 5 post partum
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, approximately 18 hours before blood sampling but allowed access to water ad libitum
- How many animals: 5 animals/sex/dose
- Parameters checked in table 5.7.1/2.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were obtained on the day before or on the day of the scheduled necropsy from
5 males from each group. Blood samples from 5 lactating females from each group were obtained on day 5 post partum
- Animals fasted: Yes, approximately 18 hours before blood sampling but allowed access to water ad libitum
- How many animals:5 animals/sex/dose
- Parameters checked in table 5.7.1/2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: males shortly before the scheduled sacrifice and females on day 3 or 4 post partum
- Dose groups that were examined: five P generation males and five P generation females from each group (erroneously six females were investigated in group 4)
- Battery of functions tested:
a) Cage-side observations: unusual body movements (e.g. tremors, convulsions), abnormal behaviour (e.g. circling, stereotypy) and posture as well as resistance to removal.
b) Hand-held observations: palpebral closure, pinna reflex, lacrimation, pupil size, pupil reactivity, salivation, muscle tone, extensor thrust response, righting reflex and reaction to handling.
c) Open field observations: level of ambulatory activity including rearing (one minute evaluation), responsiveness to sharp noise, paw pinch, gait evaluation, quantity of urine and fecal pellets voided.
d) Categorical observations (can be made any time during the FOB): hair coat, behavior, respiration, muscle movements, eyes, hearing ability (Preyer’s reflex), urine or feces, soiling, general abnormalities, posture.
e) Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.
Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes.
Sacrifice and pathology:
Males were sacrificed after treatment of at least 28 days, when no longer needed for the assessment of reproductive effects. Pups were sacrificed on day 4 post partum. Dams were sacrificed on day 5 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.
GROSS PATHOLOGY: Yes (see table 7.5.1/3)
HISTOPATHOLOGY: Yes (see table 7.5.1/3)
Other examinations:
The testes were stained by PAS hematoxylin for qualitative sperm staging.
Statistics:
The following statistical methods were used to analyse food consumption, body weights and reproduction data:
- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied to the macroscopical findings.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
All males and females treated with 400 or 800 mg/kg bw/d pushed their head through the bedding after application from day 14 of the pre-pairing period onwards. This was considered to be a sign of discomfort following treatment rather than a toxic effect of the test item. One male and one female treated with 800 mg/kg bw/d had salivation for isolated days. In addition, one female had salivation for most of the pre-pairing, pairing and gestation periods. This was considered to be a sign of discomfort following the treatment.
Other clinical signs noted were a wound on the shoulder of one male in low-dose group and hair loss in one dam in the control group. These findings were considered to be incidental.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- Males: In high-dose group, body weight gain was statistically significantly reduced on days 3, 6 and 10 of the pre-pairing period and on the last day of the after pairing period. However, mean body weight was similar in all dose groups throughout the study. No test item-related effects were noted in the other groups.
- Females: In high-dose group 4, mean body weight gain was statistically significantly reduced over days 2 - 7 of the pre-pairing period and absolute body weight on days 3, 4 and 6 during the pre-pairing period. This was considered to be a result of treatment with the test item. No other test item-related effects were noted during the study at any dose level.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Normal responses to the taste of fragrances

- Males: Mean food consumption was very slightly reduced in medium- and high-dose groups during the first week of the pre-pairing period (-5.6% and -6.1%, respectively). Thereafter, food consumption was not affected by treatment with the test item in any dose group.
- Females: In high-dose group, mean food consumption was statistically significantly reduced over the first week of the pre-pairing period (-18.4% compared to the control group). This reduction was considered to be a test item-related effect. The statistically significant increase in low-dose group in the first week of the pre-pairing period was considered to be incidental due to the lack of a dose-dependent pattern.
No other test item-related effects were noted in the food consumption during the study at any dose level.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant increased platelets counts in medium- and high-dose males; prothrombin time outside the historical control data in high-dose females. Not adverse

- Males The level of platelets was statistically significantly increased in medium-dose groups (+23.2%, compared to the control group) and 4 (+33.8%) and was outside the historical control data in high-dose group.
- Females: In the females in high-dose group, although the prothrombin time was not statistically significantly increased (+15.3%), the level was outside the historical control data.
All other values noted for the males and females, including statistically significant changes, were within the range of the historical control data.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Increased cholesterol and globulin level in medium- and high-dose animals; increased globulin levels in medium- and high-dose males. Not adverse.

- Males The level of cholesterol was statistically significantly increased in medium- and high-dose dose groups (+28.5% and +44.0%, respectively). Both these values were outside the range of the historical control data. The level of protein (+5.3% in medium-dose group and +8.6% in high-dose group) as well as globulin (+9.6% in medium-dose group and +16.5% in high-dose group) were increased and although the values were not statistically significant, they were outside the range of the historical control data.
- Females: The level of cholesterol in medium- and high-dose groups (+59.0% and +125.5%, respectively) was outside the range of the historical control data and was statistically significantly increased in high-dose group. The level of globulin was statistically significantly increased in medium- and high-dose groups (+17.3% and +16.1%, respectively) and both levels were outside the range of the historical control data.
All other values noted for the males and females, including statistically significant changes, were within the range of the historical control data.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Reduced number of rearing in high-dose males. Dose-dependent decrease in body temperature.

In high-dose group, there was an increased incidence in males of a reduced number of rearings (80%) compared to the control group (20%). In addition, body temperature was statistically significantly decreased in males (37.9 °C compared to the control group 38.5 °C) and females (38.3 °C compared to 39.0 °C in the control group).
In medium-dose group, the body temperature of the females was statistically significantly reduced (38.5 °C compared to 39.0 °C in the control group).
Although the body temperatures were within the range of the historical control data, since they decreased in a dose-dependent manner, it may be a slight effect of the test item.
No other test item-related effects were noted.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The weights and ratios of the liver were statistically significantly increased in males and females in the groups receiving the test item. In medium- and high-dose groups, this corresponded to histopathological findings. In low-dose group, since no test item-related histopathological findings were noted and the mean absolute weight was within the historical control data, the increase was considered to be incidental.
The weights and organ/body weight ratios of the kidney were statistically significantly increased in high-dose group in the females. Since this did not correspond to any histopathological findings and the mean absolute weight was within the historical control data, it was therefore considered to be incidental.
Gross pathological findings:
no effects observed
Description (incidence and severity):
2 females in high-dose group had an enlarged liver. No other test item-related findings were noted for males or females in any group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
In the liver, there was centrilobular hepatocellular hypertrophy in males and females in medium- and high-dose groups. There was no further lesion along with this hypertrophy and therefore, an adaptive change in liver metabolism was concluded.
In the thyroid glands, there was diffuse follicular hypertrophy in some high dose males and females at minor degrees of severity. This was considered to be the result of increased metabolism in the liver. The changes in the liver and in the thyroid glands were not considered to be adverse.
In the kidneys of males in medium- and high-dose groups, there were increased degrees of severity of hyaline inclusions along with an increased incidence and severity of tubular basophilia. It is considered that the hyaline inclusions represent alpha-2-microglobulin and the increased deposition is a consequence of increased liver metabolism. The nature of this lesion may be considered to be adverse in male rats due to the increased tubular basophilia that represent regeneration from a previous tubulopathy.
In low-dose group, no test item-related findings were noted.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
800 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The induced hyaline droplet nephropathy in male rats is known to be a rat-specific lesion and is not relevant for human
Key result
Critical effects observed:
no

Discussion:

At 800 mg/kg bw/day, salivation was noted in isolated individuals. There was an increased incidence of a decreased number of rearings in the males in group 4 compared to the control group. In addition, body temperature was statistically significantly decreased in males and females in group 4. Although this was within the range of the historical control data, since body temperature decreased in a dose-dependent manner, it may be a slight effect of the test item. Mean food consumption and body weight gain were reduced slightly and transiently in the males and females in the first week of the pre-pairing period. Clinical laboratory investigations revealed that the level of platelets was statistically significantly increased in the males. In the females, the prothrombin time was outside the range of the historical control data. In both males and females, the levels of cholesterol and globulin were outside the range of the historical control data. The level of protein in the males was also outside the range of the historical control data. At necropsy, the weights and ratios of the liver were statistically significantly increased in males and females. From the macroscopical examination, two females had an enlarged liver. Microscopically, in the liver there was centrilobular hepatocellular hypertrophy and, as a result, diffuse follicular hypertrophy in the thyroid glands in males and females at minor degrees of severity. These findings were not considered to be adverse. In the kidneys of males, there was an increased degree of severity of hyaline inclusions along with an increased incidence and severity of tubular basophilia. It is considered that the hyaline inclusions represent α2-microglobulin and that the increased deposition is a consequence of increased liver metabolism. The nature of this lesion may be considered to be adverse in rat males due to the increased tubular basophilia that represent regeneration from a previous tubulopathy.

At 400 mg/kg bw/day, mean food consumption was very slightly reduced in the males during the first week of the pre-pairing period. This had no effect on the mean absolute body weights. The level of platelets was statistically significantly increased in the males. In both males and females, the levels of cholesterol and globulin were outside the range of the historical control data. The level of protein in the males was also outside the range of the historical control data. At necropsy, the weights and ratios of the liver were statistically significantly increased in males and females. Microscopical examination revealed centrilobular hepatocellular hypertrophy in males and females. In addition, in the kidneys of the males, there were increased degrees of severity of hyaline inclusions along with an increased incidence and severity of tubular basophilia. The changes that were observed for platelets may be related to changed renal function and increased excretion. Biochemical data reveal also an affection of the female kidneys, by changes in protein and ion parameters. The cause of increased globulin levels remains unclear. Other biochemical changes recorded, including cholesterol and bilirubin are likely due to changes in the liver metabolism.

At 100 mg/kg bw/d, no test item-related effects were noted in the in-life phase. No effects were noted in the clinical laboratory investigations. At necropsy, no test item-related macroscopical or microscopical findings were noted.

Conclusions:
Based on the results of the study and since the induced hyaline droplet nephropathy in male rats is known to be a rat-specific lesion and not relevant for human, the general NOAEL was considered to be 800 mg/kg bw/day.
Executive summary:

In a combined repeated dose toxicity study with the reproduction/development toxicity screening test performed in accordance with OECD test guideline No. 422 and in compliance with GLP, the test substance diluted in corn oil was administered to 10 HanRcc: WIST(SPF) rats/sex/dose by gavage at dose levels of 0, 100, 400 and 800 mg/kg bw/day. Control rats were given the vehicle alone. The test substance was administered to male rats for at least 28 days and to female rats for 16 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.


 


The following results were obtained:


- Mortality and General tolerability


All males and females in medium- and high dose groups pushed their head through the bedding after application from day 14 of the pre-pairing period onwards. One male and one female in high-dose group had salivation for isolated days. In addition, one female had salivation for most of the pre-pairing, pairing and gestation periods. These findings were considered to be a sign of discomfort following the treatment.


- Functional Observational Battery


Body temperature was statistically significantly decreased in high-dose group in males and females as well as in the females in medium-dose group. Body temperature decreased in a dose-dependent manner. However, since these changes were within the range of the historical control data, they were considered of no toxicological importance.


There was an increased incidence of a reduced number of rearings in high-dose group compared to the control group. However, in general, when only one FOB measurement is affected, the results are not considered evidence of a neurotoxic effect, mostly when the effects occurred at the high dose, in the presence of sign of toxicity such as body weigh or body temperature descreases (Guidelines for Neurotoxicity Risk Assessment, EPA, April 1998).


- Food consumption


In the males, mean food consumption was very slightly reduced in medium- and high-dose groups during the first week of the pre-pairing period. In the females, it was statistically significantly reduced in high-dose group over the first week of the pre-pairing period. These variations are normal responses to the taste of fragrances, like ST 10 C 08.


- Body weights


In high-dose group in the males, body weight gain was statistically significantly reduced on isolated days during the pre-pairing period and on the last day of the after pairing period. In the females, mean body weight gain was statistically significantly reduced over days 2 - 7 of the pre-pairing period and absolute body weight was occasionally statistically significantly reduced. These findings were correlated to lower mean food consumption and were fully reversible; therefore they are not considered as toxicologically significant.


- Clinical Laboratory Investigations


Haematology: The level of platelets was statistically significantly increased in the males in medium- and high-dose groups (+23.2 and +33.8%, respectively) and was outside the historical control data in high-dose group. In the females in high-dose group, although the prothrombin time was not statistically significantly increased, the level was outside the range of the historical control data.


Clinical biochemistry: In both males and females, the levels of cholesterol and globulin in medium- and high-dose groups were outside the range of the historical control data. The level of protein in the males in the same groups was outside the range of the historical control data.


These changes were considered as adaptive in nature, and of no toxicological concern.


- Organ Weights


The weights and ratios of the liver were statistically significantly increased in males and females in medium- and high-dose groups.


- Macroscopical Findings and Histopathological Examinations


At macroscopical examination, two females in high-dose group were noted to have an enlarged liver.


In this dose group, in the liver there was centrilobular hepatocellular hypertrophy and, as a result, diffuse follicular hypertrophy in the thyroid glands in males and females at minor degrees of severity.


These findings were considered to be adaptive responses to the test material and not to be adverse. In the kidneys of males, there were increased degrees of severity of hyaline inclusions along with an increased incidence and severity of tubular basophilia. It is considered that the hyaline inclusions represent α2-microglobulin. It may be considered that the increased deposition is a consequence of increased liver metabolism. The nature of this lesion may be considered to be adverse in rat males due to the increased tubular basophilia that represent regeneration from a previous tubulopathy.


In medium-dose group, there was centrilobular hepatocellular hypertrophy in males and females. In addition, in the kidneys of the males, there were increased degrees of severity of hyaline inclusions along with an increased incidence and severity of tubular basophilia.


 


Based on the results of the study and since the induced hyaline droplet nephropathy in male rats is known to be a rat-specific lesion and not relevant for human, the general NOAEL was considered to be 800 mg/kg bw/day.


 


Based on the results of this study, the test substance is not classified for damage to organs through prolonged oral repeated exposure according to the criteria of the Annex I of the Regulation (EC) No. 1272/2008 (CLP).


This study is considered as acceptable and satisfies the requirement for sub-acute oral toxicity endpoint.

Data source

Reference
Reference Type:
other: Draft report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
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

1
Chemical structure
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
Chemical structure
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-Tetramethyldodecahydronaphtho[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 / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Test item
Dose / conc.:
400 mg/kg bw/day (nominal)
Remarks:
Test item
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).