Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 January to 12 September 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 407 without any deviation.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3050 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on 05-07 February 2014/ signed on 24 April 2014)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Clear colorless liquid
- Storage Conditions: ca. 4 °C in the dark under nitrogen
Specific details on test material used for the study:
- Purity test date: 14 January 2014

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
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 CD strain was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 42-48 days
- Weight at study initiation: Males: 190-263 g; Females: 164-208 g
- Housing: Five animals/sex/cage (main study and recovery) in polycarbonate cages with a stainless steel mesh lid
- Diet: Rat and Mouse No. 1 Maintenance Diet (Special Diet Services), ad libitum; Non-restricted (removed overnight before blood sampling for haematology or blood chemistry and during the period of urine collection).
- Water: Potable water from the public supply, ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: 23 January to 12 September 2014

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral gavage route of administration was chosen to simulate a potential route of accidental exposure.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Method of preparation: A series of clear solutions at the required concentrations were prepared by dilution of individual weighings of the test substance.
Frequency of preparation: Weekly
Storage of preparation: Refrigerated (nominally 4 °C).

VEHICLE
- Concentration in vehicle: 6, 60 and 200 mg/L
- Amount of vehicle: 5 mL/kg bw/day

STABILITY AND HOMOGENEITY: Before commencement of treatment, the suitability of the proposed mixing procedures was determined by analysing specimen formulations at concentrations of 1 and 200 mg/mL following storage at ambient temperature (nominally 21 °C) for 24 hours and refrigerated (2-8 °C) for up to 15 days.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation prepared for administration in Weeks 1 and 4 of treatment were analysed for achieved concentration of the test substance.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily at approximately the same time each day.
Doses / concentrationsopen allclose all
Dose / conc.:
30 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:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In the preliminary study, administration of ST11 C13 to CD rats by daily oral gavage administration for 7 days at dose levels of 250, 500 or 1000 mg/kg bw/day was generally well tolerated with no significant clinical signs or any treatment-related deaths. Salivation, which was observed shortly after dosing, predominantly for animals receiving 1000 mg/kg bw/day is commonly encountered in studies where the test substance is administered orally and is not considered toxicologically significant. The chin rubbing was considered to be secondary to the excessive salivation. Low body weight gain observed for females receiving 1000 mg/kg bw/day may have been a reflection of low food consumption for these animals. None of the findings observed preclude administration of dosages up to 1000 mg/kg bw/day in the 28-day study (HLS Study No. HIK0030). Suitable doses for the 28-day study are, therefore, considered to be 1000 mg/kg bw/day (the limit dose) for the high dose and 30 and 300 mg/kg bw/day as the low and intermediate doses based on the GHS labelling points.
- Rationale for animal assignment: Randomly allocated on arrival.
- Post-exposure recovery period in satellite groups: 14 days
Positive control:
None

Examinations

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. During the acclimatisation and recovery periods, observations of the animals and their cages were recorded at least once per day.
A viability check was performed near the start and end of each working day.
One female animal was found dead on Day 26 of treatment and one female failed to recover from the anaesthesia at routine blood sampling on Day 29 of treatment.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily during Week 1 of treatment, detailed observations were recorded at the following times in relation to dose administration:
Pre-dose observation; at the end of each dosing group; one to two hours after completion of dosing all groups; as late as possible in the working day.
Daily from Day 8 of treatment until termination, detailed observations were recorded at the following times in relation to dose administration: Predose observation; one to two hours after completion of dosing of all groups

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded one week before treatment commenced (Day P1), on the day that treatment commenced (Day 1), 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 (Week P1) 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: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 29 (all main study animals); Recovery Day 15 (all recovery phase animals); Blood sampling was performed on the morning after overnight collection of urine.
- Anaesthetic used for blood collection: Yes; light general anaesthesia induced by isoflurane
- Animals fasted: Yes; Animals were deprived of food and water overnight but were allowed access to water for a minimum period of one hour prior to the commencement of blood sampling procedures.
- Parameters checked:
Haematology: Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyser: Haematocrit (Hct), Haemoglobin concentration (Hb), Erythrocyte count (RBC), Absolute reticulocyte count (Retic), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), Red cell distribution width (RDW), Total leucocyte count (WBC), Differential leucocyte count: Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M) and Large unstained cells (LUC), Platelet count (Plt), Morphology: Anisocytosis, Microcytosis, Macrocytosis, Hypochromasia and Hyperchromasia
Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using an ACL series analyser and appropriate reagent in respect of: Prothrombin time (PT) and Activated partial thromboplastin time (APTT)
Clinical chemistry: Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyser in respect of: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transferase (gGT), Total bilirubin (Bili), Total bile acids (Bi Ac), Urea, Urea Nitrogen (BUN), Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot), Albumin (Alb).
Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analysed albumin concentration.

URINALYSIS: Yes
- Time schedule for collection of urine: Day 28-29 (all main study animals); Recovery Day 14-15 (all recovery study animals)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes; Animals were placed in an individual metabolism cage overnight, without food or water (ca 16 h).
- Parameters checked:
Using manual methods: Clarity and Colour - by visual assessment, Volume - using a measuring cylinder, pH - using a pH meter, Specific gravity - by direct refractometry using a SG meter
Using Multistix reagent strips, interpreted using a Clinitek®500 instrument: Ketones, Bilirubin/bile pigments, Urobilinogen, Blood pigments
Using a Roche P Modular analyser: Protein, Creatinine, Glucose, Sodium, Potassium, Chloride

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Before treatment commenced and during each week of treatment and recovery, 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 (before dosing during the treatment period), by an observer unaware of the experimental group identities.
- Battery of functions tested: sensory activity / grip strength / motor activity
Sensory reactivity and grip strength: Sensory reactivity and grip strength assessments were performed (before dosing) on all Main study animals in Groups 2 and 3 and all Recovery phase animals during Week 4 of treatment. In addition, all recovery phase animals were tested during Week 2 of recovery.
Motor activity: During Week 4 of treatment (before dosing), the motor activity of all Main study animals in Groups 2 and 3 and all Recovery phase animals was measured using a Rodent Activity Monitoring System (Version 2.0.5), with hardware supplied by Pearson Technical Services and software developed and maintained by Huntingdon Life Sciences. In addition, all recovery phase animals were tested during Week 2 of recovery.

IMMUNOLOGY: No
Sacrifice and pathology:
SACRIFICE: Animals were killed by carbon dioxide asphyxiation with subsequent exsanguination. Main study animals were killed following 28 days of treatment, on Day 29 of the study. Recovery animals were killed following 28 days of treatment and 14 days of recovery on Day 43 of the study (Day 15 of recovery). All Main phase and Recovery phase animals were subject to a detailed necropsy.

GROSS PATHOLOGY: Yes; 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. The requisite organs were weighed for Main phase and Recovery phase animals killed at scheduled termination.

HISTOPATHOLOGY: Yes
Fixation: Tissues were routinely preserved in 10 % Neutral Buffered Formalin with the exception of those detailed below:
Testes: In modified Davidson’s fluid; Eyes: 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. Main study animals of Groups 1 and 4 killed at a scheduled interval.
Abnormalities and liver only: All animals
Routine staining: Sections were stained with haematoxylin and eosin.
Light microscopy: Tissues preserved for examination were examined as follows:
Premature deaths: All animals from all groups
Scheduled kill:
Main study: All animals of Groups 1 and 4 (all specified in Table 7.5.1/1)
All animals of Groups 2 and 3 (Liver and abnormalities only)
Recovery: All animals of Groups 1 and 4 (Liver and abnormalities only)
Other examinations:
None
Statistics:
See "Any other information on materials and methods incl. tables"

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Signs considered attributable to administration of ST11 C13 at 1000 mg/kg bw/day comprised reddening of the ears for all animals on Days 2 and approximately half the animals on Day 3 from 1-2 hours after dosing until the end of the working day. This sign was also apparent, though at a lower incidence, on most days in Week 2 at 1-2 hours after dosing for animals receiving 1000 mg/kg bw/day. Reddening of the limbs and muzzle was also recorded on Day 12 for one male receiving 1000 mg/kg bw/day. Reddening was not observed during Weeks 3 or 4 of treatment or during the recovery period.
- In addition, abnormal (swaying) gait was observed at 1 to 2 hours after dosing on Day 2 for one male receiving 1000 mg/kg bw/day. This was still present at the end of the working day, but was no longer apparent the following morning and was not observed on subsequent days.
- Salivation was observed shortly after dosing during Week 1 for animals receiving 1000 mg/kg bw/day and one male receiving 300 mg/kg bw/day, with associated chin rubbing in a small number of these animals. These signs were absent 1 to 2 hours after dosing. These clinical signs are considered to be typically related to the taste of the test-material and not of toxicological importance.
- The general appearance and behaviour of the animals during the detailed physical examination and the arena observations were not affected by treatment.
CONCLUSION : Non-adverse effects / reversible within the recovery period
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- There were no deaths that were attributed to treatment.
- A control female was found dead on Day 26. No clinical signs were observed prior to death and there no significant macroscopic or microscopic findings. The cause of death is unknown, however, as this is a control animal death was not attributable to treatment.
- A female receiving 30 mg/kg bw/day died during routine blood sampling on Day 29 when it failed to recover from the anaesthesia. There were no preceding clinical signs and no significant macroscopic or microscopic findings for this animal and, therefore, this death was considered incidental and unrelated to treatment.
CONCLUSION : Non-adverse effects
Body weight and weight changes:
no effects observed
Description (incidence and severity):
- Body weight gain for females receiving 1000 mg/kg bw/day was slightly low during Week 1 when compared with the controls with statistical significance. This trend was not apparent from Week 2 onwards, with body weight gains for these animals being slightly higher or similar to those of the Controls resulting in a similar overall value.
- Body weight gain for males at 1000 mg/kg bw/day and animals receiving 30 or 300 mg/kg bw/day was unaffected by treatment.
- Bodyweight gain during the recovery period was similar for control and previously treated animals.
CONCLUSION : Non-adverse effects / reversible
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was not affected by treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
- The visual assessment of water intake did not reveal any clear treatment-related effect and, consequently, quantitative measurements were not performed.
- A visual assessment of water intake indicated apparently higher water consumption than the controls on Day 13 for animals receiving 1000 mg/kg bw/day. Since no further differences were seen throughout the study, this isolated finding was considered to have occurred by chance.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
- The haematological examination after 28 days of treatment revealed low haematocrit, haemoglobin concentration and erythrocyte count and high mean cell haemoglobin and mean cell haemoglobin concentration for females receiving 1000 mg/kg bw/day. There was also a decrease in reticulocyte count in these animals, though this did not attain statistical significance. The only similar effect in males was slightly high mean cell haemoglobin concentration at 1000 mg/kg bw /day. Large unstained cell counts were higher than those of the controls for males receiving 1000 mg/kg bw /day and prothrombin time was increased for females receiving 1000 mg/kg bw /day.
- Some statistically significant differences were reported at the end of the recovery period for parameters that were unaffected during the treatment period and these were considered of no biological or toxicological significance. These included low platelet counts and increased activated partial thromboplastin times for previously treated females, the latter difference being attributed to low control values.
The changes in red blood cell parameters seen predominantly in females receiving 1000 mg/kg/day (low haematocrit, haemoglobin concentrations, red blood cell and reticulocyte counts and slightly high mean cell haemoglobin and mean cell haemoglobin concentration) suggest a potential effect on the haemopoietic system. There were, however, no associated pathological findings, for example, in the bone marrow or spleen. The relatively small changes in these parameters may represent low level toxicity in these animals but were considered not to be adverse at the magnitude of change observed.
CONCLUSION : Non-adverse effects / reversible within the recovery period
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- The biochemical examination of the blood plasma obtained after 28 days of treatment revealed low glucose and chloride concentrations and high triglyceride and phosphorus concentrations for animals receiving 1000 mg/kg bw/day. In addition, creatinine concentration was high and bile acid concentration was low for females receiving 1000 mg/kg bw/day. Calcium concentration was low for all treated groups, with no dose-relationship.
- On completion of the 14 day recovery period, the above changes were no longer apparent, with the exception that calcium concentration was still statistically significantly low when compared with the controls for previously treated males. The majority of these values were, however, within the background range.
- Some statistically significant differences were reported at the end of the recovery period for parameters that were unaffected during the treatment period and these were considered of no biological or toxicological significance. These included slightly low total protein with an associated high albumin/globulin ratio for previously treated males and low alanine amino-transferase activity for previously treated females.
CONCLUSION : Non-adverse effects / partially reversible within the recovery period / no biological or toxicological significance
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
- Urinalysis investigations after 28 days of treatment revealed low pH for animals receiving 300 or 1000 mg/kg bw/day. A higher incidence of ketones was also present in the urine of males receiving 1000 mg/kg bw/day. These findings were no longer apparent after two weeks of recovery.
- Slightly high glucose, sodium and chloride outputs for males receiving 1000 mg/kg bw/day were considered directly related to slightly increased urinary volume in these animals, with the majority of individual values within the background range and therefore of no biological importance.
- Some statistically significant differences were reported at the end of the recovery period for parameters that were unaffected during the treatment period and these were considered of no biological or toxicological importance. These included slightly high specific gravity and low sodium output for previously treated females, which were considered directly related to reduced urinary volume in these animals and therefore of no biological importance.
CONCLUSION : Non-adverse effects / partially reversible within the recovery period / no biological or toxicological significance
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
- Sensory reactivity and grip strength: During Week 4 of treatment, the group mean forelimb grip strength values for males receiving 1000 mg/kg bw/day were slightly low compared to controls, attaining statistical significance. All the group mean forelimb and hindlimb grip strength scores were, however, within the historical control range and consequently this was considered unlikely to be an effect of treatment with ST11 C13.
During Week 2 of recovery, group mean forelimb grip strength for animals previously treated at 1000 mg/kg bw/day was similar to that of the controls.
There were no effects of treatment apparent on sensory reactivity data.
- Motor activity: Motor activity assessment in Week 4 revealed that high beam scores for all treated female groups and the majority of the group mean low beam scores for females receiving 300 or 1000 mg/kg bw/day were low compared with Controls, with the total scores showing a dose-relationship. The majority of the scores were, however, within the historical control data range and, therefore, the differences from controls were not clearly attributable to treatment and not of a magnitude to be adverse.
CONCLUSION : Non-adverse effects / within historical data range
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- High absolute and body weight-adjusted liver and kidney weights for males and females at 1000 mg/kg bw/day and for males at 300 mg/kg bw/day (maximum effect for each tissue: 1.2X control for body weight- adjusted weights); attaining statistical significance for liver and kidney weights).
- Absolute and bodyweight-adjusted heart weights were also high, compared with controls, for males which received 1000 mg/kg bw/day (maximum effect 1.2X control); attaining statistical significance. There was no similar effect in females. The change in heart weight in males given 1000 mg/kg/day was not associated with any histopathological change and was, therefore considered of no toxicological importance.
- For animals killed on completion of the recovery period, liver weights for previously treated animals were similar to those of the controls indicating full recovery had occurred. Kidney weights for previously treated females were still slightly higher than those of the concurrent controls, attaining statistical significance, but they were lower than those observed after four weeks of treatment at this dose level, indicating at least partial recovery had occurred. Kidney weights for previously treated males had recovered fully.
- The reduced thymus weights observed at the end of the recovery period for previously treated males were considered incidental since no similar effect was apparent at the end of the treatment period.
CONCLUSION : Non-adverse effects / partially reversible within the recovery period / no biological or toxicological significance
Gross pathological findings:
no effects observed
Description (incidence and severity):
- The macroscopic examinations performed on completion of the treatment and recovery periods revealed no test substance related lesions.
- The incidence and distribution of all findings were consistent with the commonly seen background of macroscopic findings in CD rats at Huntingdon Life Sciences laboratories.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Animals killed after 4 weeks of treatment
- Treatment related findings: Changes attributed to treatment with ST11 C13 were seen in the liver.
Liver: Minimal to slight vacuolation was seen in the cytoplasm of periportal liver cells in most of the animals treated at 1000 mg/kg bw/day in both sexes. Sporadic incidences of this finding were noted in other groups, including one male control, and are likely to be related to the corn oil vehicle. Minimal vacuolation in two males treated with 300 mg/kg bw/day was considered not to be treatment-related due to its low incidence and severity.
- Incidental findings: All other histological changes were similar across control and treated groups, and were considered to be unrelated to treatment.

Animals killed after 2 weeks of recovery
- The microscopic examination performed after 2 weeks of recovery revealed the following changes in the liver:
Liver: Minimal vacuolation was seen in the cytoplasm of periportal liver cells in one male control animal and in one male and one female animal previously treated at 1000 mg/kg bw/day. This was considered to represent background levels of the finding. Treatment related vacuolation was considered to have recovered.
- All other findings were similar to those in controls and were considered to be unrelated to treatment.

CONCLUSION : Non-adverse effects / reversible within the recovery period
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: All the changes were shown to be reversible during a subsequent two week period without treatment and none of the findings were considered adverse

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

The dosage of 1000 mg/kg/day was well tolerated with minimal clinical signs comprising transient reddening of the ears, and for one animal, reddening of the limbs and muzzle on Day 12 and abnormal (swaying) gait on Day 2 for one male. Although abnormal gait was observed for just one animal (No. 27), it cannot be discounted that this was related to treatment. Salivation and chin rubbing, which were observed shortly after dosing during Week 1 are frequently observed signs in studies where the test material is administered by gavage and are considered to be typically related to the taste of the test-substance and not considered of toxicological importance.

Histopathological examination identified the liver as a target organ. Microvesicular vacuolation was seen in the cytoplasm of periportal liver cells at a minimal severity in males treated at 1000 mg/kg/day and a minimal to slight severity in females treated at 1000 mg/kg/day. Minimal vacuolation, seen in two males given 300 mg/kg/day, was considered unlikely to be treatment-related at this incidence and severity, particularly as there were no corresponding clinical chemistry changes. Oil red O fat staining confirmed that the microvesicular vacuolation was associated with increased fat in hepatocytes and correlated with statistical significant higher relative liver weight values. It is also related to the high plasma triglyceride levels in both sexes and the raised urinary ketone levels seen in animals of the same group. The lengthened prothrombin times seen for females receiving 1000 mg/kg/day at the end of the treatment period were also attributed to the effect of the liver since the liver is the site of the biosynthesis of several clotting factors. All these changes in the liver were considered to have recovered at the end of the two week recovery period. In the absence of any evidence of hepatocellular damage or necrosis, microvesicular vacuolation is likely to indicate increased storage of fat, and appears when there is accelerated mobilization of lipid from adipose tissue (Haschek et al, 2013). This alteration in lipid metabolism may be secondary to the lower blood glucose levels seen at 1000 mg/kg/day.

Findings indicative of an effect on the kidney comprised increased kidney weights at 1000 mg/kg/day in both sexes and at 300 mg/kg/day in males, increased plasma creatinine in females receiving 1000 mg/kg/day, though this was not associated with any increase in urea levels, some minor effects (increases) in some urinary electrolytes (Na and Cl) and glucose, a slight increase in plasma phosphorus and low plasma chloride concentration and low pH. The slightly high glucose, sodium and chloride outputs were considered directly related to slightly increased urinary volume in these animals, with the majority of individual values within the background range and therefore of no biological importance. There were no associated renal pathology changes, no change in water consumption and all the changes resolved following cessation of treatment. Therefore, these changes were considered to be adaptive, possibly in response to the excretion of the test substance.

The significance of the low plasma calcium concentration seen in all treated groups of both sexes, but without dose-relationship is of unclear relationship to treatment. It may be associated with the low urinary pH, or with the adaptive change on the kidney. The majority of individual values were within the HLS background range and, therefore, considered not of toxicological importance.

The changes in red blood cell parameters seen predominantly in females receiving 1000 mg/kg/day (low haematocrit, haemoglobin concentrations, red blood cell and reticulocyte counts and slightly high mean cell haemoglobin and mean cell haemoglobin concentration) suggest a potential effect on the haemopoietic system. There were, however, no associated pathological findings, for example, in the bone marrow or spleen. The relatively small changes in these parameters may represent low level toxicity in these animals but were considered not to be adverse at the magnitude of change observed.

The low bodyweight gain observed during the first week of treatment for females receiving 1000 mg/kg/day, in the absence of any associated reduction in food consumption, was considered to indicate non-specific toxicity. The effect was not apparent from Week 2 onwards, indicating an amelioration of effect.

During Week 4 of treatment, lower than control motor activity (rearing and cage floor activity) was recorded for all treated female groups with the total scores showing a dose-relationship. However, only two of the 6-minute intervals attained statistical significance, and the majority of the group mean 6-minute interval scores and the group mean total scores were within the historical control data range, neither was there any effect in males. The differences from controls were, therefore, not clearly attributable to treatment and not of a magnitude to be adverse.

The change in heart weight in males given 1000 mg/kg/day was not associated with any histopathological change and was, therefore considered of no toxicological importance.

Administration of 1000 mg/kg/day was associated with transient clinical signs and minor disturbance of bodyweight, clinical pathology parameters and organ weight change and microvesicular vacuolation in the liver. Vacuolation of the liver, with associated increases in liver weight, plasma triglyceride levels and increases in urinary ketones, indicate accelerated mobilisation of lipids from adipose tissue. The vacuolation is, therefore, likely to represent increased storage of fat, secondary to altered lipid metabolism. In the absence of any hepatocellular damage, necrosis or increases in liver enzymes, the changes observed in the liver were considered not to be adverse.

As all the changes were shown to be reversible during a subsequent two week period without treatment and none of the findings were considered adverse, the dosage of 1000 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) in this study.

Applicant's summary and conclusion

Conclusions:
Under the test conditions, the NOAEL was concluded to be 1000 mg/kg bw/day in rats therefore it is not classified for damage to organs through prolonged oral repeated exposure according to the Regulation EC No. 1272/2008 (CLP) and to the GHS.
Executive summary:

In a repeated dose toxicity study performed in accordance with OECD test guideline No. 407 and in compliance with GLP, test item ST 11 C 13 was administered by gavage to 5 male and 5 female Crl:CD(SD) rats at doses of 30, 300 or 1000 mg/kg bw/day for 28 consecutive days. A similarly constituted control group received the vehicle, corn oil at the volume dose of 5 mL/kg bw/day. A further five male and five female rats were assigned to each of the control and high dose groups. These animals were treated for 28 days, followed by a 14 day period without treatment to assess the potential for any treatment-related change to recover. During the study clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, water consumption (visual observation), haematology (peripheral blood), blood chemistry, urinalysis, organ weight, macropathology and histopathology investigations were undertaken.

 

Two animals died during the study; both deaths were unrelated to treatment. A control female was found dead on Day 26 and a female receiving 30 mg/kg bw/day died during routine blood sampling on Day 29.

 

The general appearance and behaviour of the animals during the detailed physical examination and the arena observations were not affected by treatment. Signs attributed to treatment with ST11 C13 comprised reddening of the ears during the first two weeks of treatment for animals receiving 1000 mg/kg bw/day and reddening of the limbs and muzzle on Day 12 for one male receiving 1000 mg/kg bw/day. In addition, abnormal (swaying) gait was observed on Day 2 of treatment at 1 to 2 hours after dosing for one male at 1000 mg/kg bw/day, persisting to the end of the working day but not seen on subsequent days. Salivation was observed shortly after dosing during Week 1 for animals receiving 1000 mg/kg bw/day and one male receiving 300 mg/kg bw/day, with associated chin rubbing in a small number of these animals.

 

Motor activity assessment in Week 4 revealed that high beam scores for all treated female groups and the majority of the group mean low beam scores for females receiving 300 or 1000 mg/kg bw/day were low compared with Controls, with the total scores showing a dose-relationship. The majority of the scores were, however, within the historical control data range and, therefore, the differences from controls were not clearly attributable to treatment and not of a magnitude to be adverse.

 

Body weight gain for females receiving 1000 mg/kg bw/day was slightly low during Week 1 only, with an overall gain (Days 1 to 28) similar to controls. Males were unaffected. Food consumption and visual water consumption were not affected by treatment.

 

The haematological examination after 28 days of treatment revealed low haematocrit, haemoglobin concentration and erythrocyte count and high mean cell haemoglobin and mean cell haemoglobin concentration for females receiving 1000 mg/kg bw/day. There was also a decrease in reticulocyte count in these animals, though this did not attain statistical significance. The only similar effect in males was slightly high mean cell haemoglobin concentration at 1000 mg/kg bw/day. Large unstained cell counts were higher than those of the controls for males receiving 1000 mg/kg bw/day and prothrombin time was increased for females receiving 1000 mg/kg bw/day.

 

The biochemical examination of the blood plasma obtained after 28 days of treatment revealed low glucose and chloride concentrations and high triglyceride and phosphorus concentrations for animals receiving 1000 mg/kg bw/day. In addition, creatinine concentration was high and bile acid concentration was low for females receiving 1000 mg/kg bw/day. Calcium concentration was low for all treated groups, with no dose-relationship.

 

Urinalysis investigations after 28 days of treatment revealed low pH for animals receiving 300 or 1000 mg/kg bw/day. A higher incidence of ketones was also present in the urine of males receiving 1000 mg/kg bw/day.

 

Analysis of organ weights for animals killed after 28 days of treatment revealed, when compared with the controls, high absolute and bodyweight-relative liver and kidney weights for males and females at 1000 mg/kg bw/day and for males at 300 mg/kg bw/day and high absolute and bodyweight-relative heart weights for males which received 1000 mg/kg bw/day.

The macroscopic examinations performed on completion of the treatment and recovery periods revealed no test substance related lesions.

 

Minimal to slight reversible microvesicular vacuolation was seen in the periportal liver cells in males and females treated at 1000 mg/kg bw/day.

 

All the above changes were no longer apparent on completion of the two week recovery period, with the exception of plasma calcium concentration which was still slightly low for previously treated males but was within normal background ranges and, therefore, not of toxicological significance.

 

Under the test conditions, the NOAEL was concluded to be 1000 mg/kg bw/day in rats.

The test material is therefore not classified for damage to organs through prolonged oral repeated exposure according to the criteria of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

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