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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 15 to December 08, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD 407 Guideline without any deviation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

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:
other: OPPTS 870.3050, Repeated Dose 28-Day Oral Toxicity Study in Rodents, July 2000.
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on March 07, 2005/ signed on June 09, 2005)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Appearance: White solid
- Storage condition of test material: Room temperature in the dark under nitrogen

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat was chosen as the test species because of its acceptance as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Crl:CD® (SD)IGS BR strain was used because of the historical control data available in this laboratory
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, Kent, England
- Age at study initiation: 41 – 45 days
- Weight at study initiation: 220-253 g (males), 162-191 g (females)
- Fasting period before study: Not applicable
- Housing: The animals were housed five of one sex per cage. The cages were made of a stainless steel body with a stainless steel mesh lid and floor, and were suspended above absorbent paper which was changed at appropriate intervals.
- Diet: Standard rodent diet (Rat and Mouse No. 1 Maintenance Diet from Special Diets Services Ltd., Witham, Essex, England), ad libitum
- Water: Potable water taken from the public supply, ad libitum
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 °C
- Humidity: 40-70 %
- Air changes: Each cage was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated.
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route of administration was chosen to simulate the conditions of potential human exposure
Vehicle:
other: 1.0% w/v methylcellulose in water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The required amount of test substance, as supplied, was ground into a fine powder in a mortar and small amounts of vehicle were then mixed with it to form a smooth paste. Further amounts of the vehicle were gradually added to the paste and mixed with it. The final suspension was mixed using a high-shear homogenizer before being transferred to final containers. All formulations were prepared freshly each week and stored at 4°C in the dark.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The tested substance is stable and forms homogeneous mixtures with vehicle.
- Concentration in vehicle: 0, 1.5, 15 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation prepared for administration in Week 1 of treatment were analysed for achieved concentration of the test substance. The concentrations of the tested substance in test formulations were analyzed during the study with High performance liquid chromatograph (HPLC) fitted with a UV-Vis detector.

Typical chromatographic conditions:
Analytical column: Symmetry C18, 3.5 μm, 150 x 4.6mm.
Column temperature: Ambient, nominally +21 ºC.
Mobile phase: Acetonitrile / water (80/20 v/v).
Flow rate: 1.0 mL/minute.
Detector wavelength: UV, 279 nm.
Injection volume: 10 µL
Approximate retention time: 2.5 min.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
Animals were dosed once each day at approximately the same time each day, seven days per week.
Doses / concentrationsopen allclose all
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
150 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: The dosages used were selected with reference to previous work where the tested substance was tolerated over a period of seven consecutive days up to 1000 mg/kg bw/day. Based on this result, the dosages of 0, 15, 150 and 1000 mg/kg bw/day were selected.
- Rationale for animal assignment (if not random): sequential allocation (1 per sex dose group) until all cages filled as designated
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): no data available
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice a day

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded one week before treatment commenced (Week -1), on the day that treatment commenced (Week 0), weekly throughout the treatment and before necropsy.

FOOD CONSUMPTION:
- 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 -1), and each week throughout the treatment. From these records the mean weekly consumption per animal (g/rat/week) was calculated for each cage.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Fluid intake was assessed by daily visual observation.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 29
- Anaesthetic used for blood collection: Isoflurane
- Animals fasted: Yes; overnight fasting
- How many animals: All animals
- Parameters checked (haematology): Haemoglobin (Hb), Erythrocyte count (RBC), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), Total white cell count (WBC), Differential WBC count - Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC), Platelet count (Plt), Prothrombin time (PT) and Activated partial thromboplastin time (APTT)
- Parameters checked (clinical chemistry): Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Sodium (Na), Potassium (K), Total protein (Total Prot), Albumin (Alb) and Albumin/globulin ratio (A/G Ratio)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: daily
- Dose groups that were examined: 5 males and 5 females from each group.
- Battery of functions tested: sensory reactivity, grip strength, motor activity

Sacrifice and pathology:
NECROPSY: All animals were killed by carbon dioxide asphyxiation at the end of the treatment period.
GROSS PATHOLOGY: Yes; all animals were subject to a detailed necropsy.
HISTOPATHOLOGY: Yes (All tissues: Control and High-dose groups)
- Testes and epididymides were fixed in Bouin’s solution prior to transfer to 70% industrial methylated spirit. Samples (or the whole) of the other tissues listed below from all animals were preserved in 10% neutral buffered formalin:
Adrenals, brain, caecum, colon, duodenum, epididymides, femurs+, head#, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes – mandibular and mesenteric, oesophagus#, ovaries, pancreas#, prostate, rectum, sciatic nerves+, seminal vesicles, spinal cord, spleen, sternum#, stomach, testes, thymus, thyroid with parathyroids, trachea, urinary bladder, uterus and cervix

+ Only one processed for examination
# Not processed for examination

Tissue samples were dehydrated, embedded in paraffin wax, sectioned at approximately four to five micron thickness and stained with haematoxylin and eosin, except the testes which were stained using a standard periodic acid/Schiff (PAS) method.
Other examinations:
ORGAN WEIGHTS: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus
Statistics:
All statistical analyses were carried out separately for males and females.

All analyses were carried out using the individual animal as the basic experimental unit.

The following data types were analyzed at each time point separately:
Grip strength and motor activity
Bodyweight, using gains over appropriate study periods
Blood chemistry and haematology
Organ weights, both absolute and adjusted for terminal bodyweight
Pathological findings, for the number of animals with and without each finding

For categorical data, including pathological findings, the proportion of animals was analysed using Fisher’s Exact test (Fisher 1973) for each treated group versus the control.
For continuous data, Bartlett’s test (Bartlett 1937) was first applied to test the homogeneity of variance between the groups. Using tests dependent on the outcome of Bartlett’s test, treated groups were then compared with the Control group, incorporating adjustment for multiple comparisons where necessary.

Significant differences between Control and treated groups were expressed at the 5% (p<0.05) or 1% (p<0.01) level.
a - p<0.05; b - p<0.01 - using categorical or parametric tests
A - p<0.05; B - p<0.01 - using non-parametric tests

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Treatment related clinical sign of underactivity was observed occasionally in males and females receiving 1000 mg/kg/day during the treatment period. This sign was generally observed on isolated occasions from approximately 1 hour to 4 hours after dosing. This time onset might indicate that underactivity resulted from a systemic effect of ST 06 C 05, leaving time for some absorption to occur first.
Mortality:
no mortality observed
Description (incidence):
There was no unscheduled deaths during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Low bodyweight gains with associated low efficiency of food utilisation observed in males treated at 1000 mg/kg/day were considered to be due to treatment. However, due to the small magnitude of difference from controls, no actual bodyweight losses being apparent and some males having overall gains that were within the concurrent control range, these low bodyweight gains were not considered to be detrimental to the overall health of the animals.
Therefore, they were considered not to represent overt toxicity within the context of this study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The food consumption over the study period for treated groups was generally similar to that of control thus indicating no effect of treatment.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Low bodyweight gains with associated low efficiency of food utilisation observed in males treated at 1000 mg/kg/day were considered to be due to treatment. However, due to the small magnitude of difference from controls, no actual bodyweight losses being apparent and some males having overall gains that were within the concurrent control range, these low bodyweight gains were not considered to be detrimental to the overall health of the animals.
Therefore, they were considered not to represent overt toxicity within the context of this study.
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Analysis for animals receiving treatment at 1000 mg/kg bw/day revealed lower mean white blood cell counts for males (reflected in lower mean eosinophil counts and lymphocyte counts) and higher mean prothrombin time for animals of both sexes, when compared with their respective control mean values. No relevant effects were recorded for other dose levels.
No histopathological findings could be linked either to the lower than control white blood cell (WBC) count observed for males or to the higher than control mean prothrombin time noted for animals of both sexes receiving treatment at 1000 mg/kg/day. Additionally, the values measured for these parameters were within expected background range (7.99 to 18.29 in males for WBC and 12.3 to 16.5 in females for prothrombin time) thus, although the changes may be attributable to treatment, they are considered to be of no toxicological importance in the context of this study.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Analysis showed that animals receiving treatment at 1000 mg/kg bw/day had lower urea concentrations and higher albumin concentrations with concomitant higher A/G ratio for animals of both sexes, as well as lower creatinine concentration for females when compared with their respective control mean values. No relevant effects were recorded for other dose levels.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
All animals were assessed for physical condition and behaviour during handling and after being placed in a standard arena and no deviation from normal was recorded.
The motor activity assessment revealed a reduced activity (both cage floor and rearing activity) in males receiving 1000 mg/kg/day. Despite these recordings occurring approximately 24 hours after dosing, there was some correlation with the occasional post-dose underactivity seen at this level (Animal number 19 was noted to be underactive after dosing on the day the motor activity was performed). The differences from controls in motor activity were minor as indicated by only being seen on a few occasions in individual animals after dosing and only being clearly evident during the first part of the 1-hour motor activity sessions, when the animals, including controls, were showing the highest activity. Although females receiving 1000 mg/kg/day were also noted to show a low level of post-dose underactivity, they did not show any clear treatment-related effects on motor activity. There was no indication that the slightly lower motor activity in males was a neurotoxic effect. It is possible that the slightly lower activity observed in males receiving 1000 mg/kg/day was due to a combination of general toxicity (as indicated by the low bodyweight gains and inefficient food utilisation) and possibly some slight abdominal discomfort due to the higher liver weights and hepatic enlargement seen in these animals. The aetiology of the low rearing activity noted in males treated at 150 mg/kg/day is not clear from this study. In the absence of any other corroborative findings at this level in males, the low activity is considered not to be of toxicological importance.
Sensory reactivity and grip strength were considered to be unaffected by treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In the liver, centrilobular hepatocyte hypertrophy was found in the majority of animals, of both sexes, receiving treatment at 1000 mg/kg/day. This treatment-related finding correlates with the reported macroscopic finding at necropsy of liver enlargement, and with the high liver weights recorded for these animals. The liver is one of the major organs involved in xenobiotic metabolism and enlargement/increased weight due to hepatocyte hypertrophy is a typical adaptive response. Therefore, the changes reported in the liver are considered not to be adverse but do indicate the liver as a target organ.
The increased weights of the kidney, recorded for females treated at 1000 mg/kg/day, might have reflected an increased efficiency of the kidneys in animals treated at 1000 mg/kg/day. A consequence of such an increased efficiency would have been to lower waste concentrations in plasma of affected animals. There was evidence of a reduction in urea and creatinine plasma concentrations, which are waste products, in the females treated at 1000 mg/kg/day and of a reduction in plasma urea concentration in the males, although no increase in the males’ kidney weights was noted. No correlated histopathology findings were observed, thus the higher weights recorded for females at 1000 mg/kg/day were not indicative of toxicity. Alternatively, the lower plasma urea concentrations might have been linked with the higher plasma albumin concentrations observed in males and females receiving 1000 mg/kg/day. As urea is a breakdown product of protein, if a reduction in the breakdown of protein (e.g. albumin) in the plasma occurred, lower plasma urea concentrations concomitant with higher plasma albumin concentration would not be unexpected.
Statistically significantly lower than control bodyweight-adjusted mean ovaries weights were recorded for all the treated female groups. There was, however, no evidence of a dose relationship and no histopathology findings were observed in the ovaries of the treated females. It was therefore concluded that these organ-weight differences were the reflection of the stage of oestrus each cage of animals was in and that these changes were not related to treatment.
Although lower than control mean adrenals and spleen weights (adjusted for bodyweight) were noted for males treated at 1000 mg/kg/day, in the absence of any histopathology findings in the adrenals or in the spleen, these changes were considered to be of no toxicological importance in the context of this study.
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
behaviour (functional findings)

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

The homogeneity and stability was confirmed for test item in 1% methylcellulose formulations at nominal concentrations of 1 mg/mL and 100 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, at ambient temperature storage for 2 days and at refrigerated storage for 8 days. The storage times represented the maximum time from preparation to completion of administration.

 

Formulation analysis showed that the mean concentrations of the test substance in dose formulations were within ±6% of nominal concentrations, confirming accurate formulation.

Applicant's summary and conclusion

Conclusions:
Under the test conditions, the NOAEL for test item is 150 mg/kg bw/day in rats. The test item is not classified according to the annex I of the Regulation (EC) No. 1272/2008 (CLP) and of the GHS.
Executive summary:

In a repeated dose oral toxicity study conducted according to the OECD Guideline 407 and in compliance with GLP, test item was administered daily by oral gavage to groups of Sprague-Dawley rats (5/sex/dose) at the dose-levels of 0 (vehicle), 15, 150 and 1000 mg/kg bw/day in the vehicle (1.0% w/v methylcellulose in water) with the dose volume of 10 mL/kg bw/day for 28 days. Examinations during the study included: clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, bodyweight, food consumption, haematology, blood chemistry, organ weight, gross pathology and histopathology

 

There were no unscheduled deaths during the study and treatment related effects observed were confined to animals receiving 1000 mg/kg bw/day. Sensory reactivity and grip strength were considered to be unaffected by treatment for all animals. Animals of both sexes receiving treatment at 1000 mg/kg bw/day were observed with low transient incidences of post-dose salivation, underactivity, abnormal gait and flat posture. Motor activity assessment revealed a slightly reduced activity (both cage floor and rearing activity) for the first half hour of the 1-hour recording period in males receiving 1000 mg/kg bw/day, whereas males at 150 mg/kg bw/day exhibited a reduced rearing activity. Low bodyweight gain and low efficiency of food utilisation, recorded in male animals receiving 1000 mg/kg bw/day, were considered to be related to treatment, whereas food consumption was considered not to be affected by treatment. At the end of the treatment period, analysis of the haematological parameters for animals receiving treatment at 1000 mg/kg bw/day revealed lower than control white blood cell count for males and higher than control mean prothrombin time for animals of both sexes. Analysis of the blood chemistry parameters, on this occasion, revealed for animals treated at 1000 mg/kg bw/day, lower than control urea concentrations and higher than control albumin concentrations with concomitant higher albumin to globulin (A/G) ratios for animals of both sexes, as well as lower creatinine concentrations for females. In males and females receiving treatment at 1000 mg/kg bw/day, higher than control mean liver weights were measured, enlargement of the liver was noted at the gross pathology examination and liver centrilobular hepatocyte hypertrophy was found in the majority of animals at the histopathology examination. Additionally, higher than control mean kidney weights were recorded for the females and lower than control mean adrenals and spleen weights were recorded for the males. Liver was identified as a target organ on this study with adaptive but not adverse treatment-related changes.

 

In summary, the only findings at 1000 mg/kg bw/day which were considered to be indicative of toxicity were slightly lower bodyweight gains and motor activity in males and occasional underactivity and abnormal gait in both sexes. Due to these findings of minor toxicity, the dose level of 1000 mg/kg bw/day could not be classed as a clear No Observed Adverse Effect Level (NOAEL). No adverse effects of treatment were observed at the preceding dose level of 150 mg/kg bw/day. A difference in male rearing behaviour (slightly low rearing activity in test versus control animals) were observed at 150 mg/kg bw/day, but in the absence of statistical significance or any other findings, this activity was considered not to be of toxicological importance. Thus 150 mg/kg bw/day can be classed as the study NOAEL.

 

Under the test conditions, the NOAEL for test item is 150 mg/kg bw/day in rats. The test item is not classified according to the annex I of the Regulation (EC) No. 1272/2008 (CLP) and of the GHS.

This study is considered as acceptable and satisfies the requirement for subacute repeated dose toxicity endpoint.