Reaction mass of (3aR,5aS,9aS,9bR)-3a,6,6,9a-Tetramethyldodecahydronaphtho[2,1-b]furan and (3aS,5aR,9aR,9bS)-3a,6,6,9a-Tetramethyldodecahydronaphtho[2,1-b]furan

Endpoint summary

Description of key information

- 90-day repeated dose toxicity study by oral gavage administration in rats: NOAEL = 100 mg/kg bw/day based on increased body weight-adjusted liver weights apparent for both sexes at 400 or 800 mg/kg bw/day (OECD 408, GLP, rel. 1).

- Combined repeated dose toxicity study with the reproduction/development toxicity screening test: NOAEL = 800 mg/kg bw/day (OECD 422, GLP, rel. 1).

 

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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).

Reference 2

Endpoint:

repeated dose toxicity: oral, other

Remarks:

range-finding study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 2008-07-14 to 2008-09-01

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Dose range-finding study

Qualifier:

no guideline required

Principles of method if other than guideline:

The purpose of this study was to select suitable dosages to be used in the subsequent combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in the Han Wistar rat.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

other: HanRcc: WIST(SPF)

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: 288 to 313 g; Females: 192 to 207 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: minimum 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: 2008-07-14 To: 2009-09-01

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): 13890412

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On the first treatment day, samples 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 (4 hours and 7 days). 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.

All concentrations measured were within the acceptance limit of ±20% of the nominal concentrations. Concentrations ranged from 91.3% to 101.5% of the nominal concentrations.

Duration of treatment / exposure:

Approximately 6 weeks

Frequency of treatment:

Daily

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

3 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on an acute toxicity study in Wistar rats.

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).

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded daily during the treatment period until termination of the study.

FOOD CONSUMPTION:
Males: Pre-pairing period days 1-4, 4-8, 8-11 and 11-14; after pairing period weekly.
Females: Pre-pairing period days 1-4, 4-8, 8-11 and 11-14; gestation days 0-7 and 7-14.
No food consumption was recorded during the pairing period.

Sacrifice and pathology:

FEMALES:
On day 14 post coitum, females were sacrificed by CO2 asphyxiation and necropsied.
For evaluation of fertility, the number and distribution of implantation sites, live or dead embryos, and early and late embryonic deaths in each uterine horn were recorded. If no implantation sites were evident, the uterus was placed in an aqueous solution of ammonium sulfide to accentuate possible haemorrhagic areas of implantation sites. The number of corpora lutea in each ovary was recorded.

MALES:
Males were sacrificed and necropsied after the pairing period had been completed, following treatment for at least 28 days. Any macroscopic abnormalities were recorded.

Other examinations:

none

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, treatment-related

Description (incidence and severity):

Sign of discomfort in high-dose group.

In high-dose group, all males and females pushed their head through the bedding after application from day 3 of the pre-pairing period onwards. This was considered to be a sign of discomfort of the treatment with the test item rather than a direct toxic effect. All males and females had ruffled fur for a few days early in the pre-pairing period. Two females also had diarrhea and were in a bad condition, one of which was found dead. During the pairing, after-pairing and gestation periods, the males and females were also noted to have salivation.
In medium-dose group, one male had ruffled fur for one day in the pre-pairing period. From the pairing period onwards, all the males pushed their head through the bedding after application. During the gestation period, all females had salivation as well as pushing their head through the bedding after application.
Other clinical signs noted were hair loss in one female in the control group and one female in medium-dose group. This was considered to be incidental.

Mortality:

mortality observed, treatment-related

Description (incidence):

One high-dose female was found dead on day 4 of the pre-pairing period. All other animals in all dose groups survived until the scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight statistically reduced in high-dose group.

- Males: In high-dose group, mean body weight gain was statistically significantly reduced for most of the pre-pairing period (+9.0% compared to +14.8% on average in the control group). This was considered to be a test item-related effect. Mean absolute body weight was correspondingly reduced but not statistically significantly. During the after-pairing period mean body weight gain was similar to that of the control group but mean absolute body weight remained reduced. In other dose-groups, mean body weight and body weight gain were not affected by treatment with the test item.
- Females: In high-dose group, mean body weight gain was statistically significantly reduced at the start of the prepairing period. Thereafter, it was not considered to have been affected by the test item. Mean absolute body weight was initially reduced and then recovered. During the gestation period, it was not affected by treatment with the test item. In other dose-groups, mean body weight and body weight gain were not affected by treatment with the test item.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Males: Over the first 4 days of the pre-pairing period, mean food consumption was statistically significantly reduced in high-dose group (-38.6% compared to the control group). This was considered to be related to the treatment with the test item. For the rest of the pre-pairing period and
throughout the after pairing period, mean food consumption was similar to that of the control group. In other dose-groups, mean food consumption was similar to that of the control group during the pre-pairing and after pairing periods.
- Females: At the start of the pre-pairing period, mean food consumption was dose-dependently reduced (-9.1% at 100 mg/kg bw/d, -17.5% at 300 mg/kg bw/d and -36.4% at 1000 mg/kg bw/d 4 compared to the control group over days 1 - 4). These reductions were not statistically significant in any dose group. This was considered to be a result of treatment with the test item. During the gestation period, mean food consumption was not affected by treatment with the test item.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No abnormal macroscopical findings were noted for any animal in any group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

- MATING PERFORMANCE AND FERTILITY
Fertility and mean pre-coital time were not affected by the treatment with the test item. One female in medium-dose group did not mate and one in high-dose group mated but was not pregnant
- IMPLANTATION RATE AND IMPLANTATION LOSS
Pre-implantation loss was statistically significantly increased in medium-dose group (21.1% of corpora lutea compared to none in the control group). This lead to a statistically significantly reduced number of implantation sites (78.9% of corpora lutea compared to 100% in the control group). This was considered to be a test item-related effect.
No test item-related effects were noted in other dose-groups in the implantation data.

Key result

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reduced food consumption and body weight gain. Pre-implantation loss.

Key result

Critical effects observed:

no

none

Conclusions:

Based on the results of this dose range-finding study, dose levels of 100, 400 and 800 mg/kg bw/day were considered applicable for the subsequent dose toxicity study with reproduction/ developmental toxicity screening test.

Executive summary:

The purpose of this study was to select suitable dosages to be used in the subsequent combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in the rat.

Four groups of 3 males and 3 females were treated by gavage with the test material once daily. Males were treated over a 14-day pre-pairing period and during the pairing period and up to one day before necropsy (for a total of at least 28 days of treatment). Females were treated throughout the pre-pairing, pairing and gestation period up to day 13 post coitum.

The following dose levels were applied:

Group 1: 0 mg/kg bw/day (control group)

Group 2: 100 mg/kg bw/day

Group 3: 300 mg/kg bw/day

Group 4: 1000 mg/kg bw/day

A standard dose volume of 5 mL/kg bw with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (corn oil).

 

The following results were obtained:

- Mortality and General Tolerability

One female in group 4 was found dead on day 4 of the pre-pairing period. All other animals in all dose groups survived until the scheduled necropsy.

In group 4, all males and females pushed their head through the bedding after application from day 3 of the pre-pairing period onwards. This was considered to be a sign of discomfort of the treatment with the test item rather than a direct toxic effect. All males and females had ruffled fur for a few days early in the pre-pairing period. Two females also had diarrhea and were in a bad condition, one of which was found dead. During the pairing and after-pairing periods, the males and females were also noted to have salivation.

In group 3, one male had ruffled fur for one day in the pre-pairing period. From the pairing period onwards, all the males pushed their head through the bedding after application. During the gestation period, all females had salivation as well as pushing their head through the bedding after application.

- Food Consumption and Body Weights

In group 4, mean food consumption and body weight gain were reduced at the start of the pre-pairing period in both males and females.

In groups 2 and 3, mean food consumption in the females was slightly reduced at the start of the pre-pairing period. This did not have any effect on body weight gain.

- Reproduction Data

Pre-implantation loss was increased and the number of implantation sites was decreased in group 4. However,the data showed that of the two pregnant females at the maximum dose level, one had normal data and the other had only one implant. Due to the small number of females used in this range-finding study, it is difficult to estimate if this observation is related to treatment or due to an abnormal female. Using a worst-case approach, the high-dose level to be used in the main study was lowered to 800 mg/kg bw/d.

- Macroscopical Findings

No abnormal macroscopical findings were noted for any animal in any group.

 

Based on the results of this dose range-finding study, dose levels of 100, 400 and 800 mg/kg bw/day were considered applicable for the subsequent dose toxicity study with reproduction/ developmental toxicity screening test.

Reference 3

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The key study is GLP-compliant and of high quality (Klimisch score = 1)

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A 90-day repeated dose toxicity study by oral administration (via gavage) was performed (Labcorp, 2022, K, rel.1) :

In this 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).

 

A dose range-finding study (Harlan, 2009, S, rel.2) to select suitable dosages to be used in the subsequent combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in the rat was performed:

In the dose range-finding study for the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 3 rats/sex/dose were treated by gavage at the dose levels of 0, 100, 300 or 1000 mg substance/kg bw/day. Males were treated over a 14-day pre-pairing period and during the pairing period and up to one day before necropsy (for a total of at least 28 days of treatment). Females were treated throughout the pre-pairing, pairing and gestation period up to day 13 post coitum. At the high dose level, reduced food consumption and body weight gain were noted. Due to pre-implantation loss in one of two females at 1000 mg/kg bw/day (see §7.8.1), the high-dose level to be used in the main study was lowered to 800 mg/kg bw/day. Due to the small number of females used in this range-finding study, it is difficult to estimate if this observation is related to treatment or due to an abnormal female. Using a worst-case approach, the high-dose level to be used in the main study was lowered to 800 mg/kg bw/day.

 

A combined repeated dose toxicity study with the reproduction/development toxicity screening test (Harlan, 2009, K, rel.1) was performed:

In the main study, considered as the key study, the substance diluted in corn oil was administered orally, by gavage at dose levels of 0, 100, 400 or 800 mg/kg bw/day, to rats according to the OECD test guideline No. 422 and in compliance with GLP.

No death occurred after the administration of the substance. Some effects of discomfort, such as salivation, were observed following treatment at the two highest dose levels. Increased liver weight, biochemical changes (bilirubin, cholesterol, protein) and liver hypertrophy were considered to be part of an adaptive response to an increase in metabolic demand. The induced hyaline droplet nephropathy observed in male rats at 800 and 400 mg/kg bw/day is known to be a rat-specific lesion and therefore is not relevant for human.

The No-Observed-Adverse-Effect-Level (NOAEL) for repeated dose toxicity is therefore 800 mg/kg bw/day.

 

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No 1272/2008.

 

Self-classification:

Based on the available data, no additional classification is proposed regarding the specific target organ toxicity after oral dose-repeated exposure acording to the Annex I of the regulation (EC) No. 1272/2008 (CLP).