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Administrative data

Description of key information

Repeated dose Toxicity: Oral route

Under the conditions of the study, the No Observed Adverse Effect Level (NOAEL) was considered to be 100 mg/kg/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 March 2016 to 17 June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3050
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Official notice of J MHLW, METI and ME (31 March 2011), YAKUSHOKUHATSU No. 0331. 7, SEIKYOKU No. 5, KANPOKIHATSU No. 110331009
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Han Wistar
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 RccHan™;WIST strain was used because of the historical control data available.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: 32 males and 32 females (pregnancy not specified). Spare animals were removed from the study room after treatment commenced.
- Age at study initiation: 37 to 43 days.
- Weight at study initiation: The males weighed between 117 to 153 g and the females weighed between 103 to 139 g.
- Fasting period before study: Not specified
- Housing: Five animals of the same sex (main study and recovery phases) per cage were housed in a polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals. The animals had access to wood based bedding which was changed at appropriate intervals each week. Males and females were blocked by sex and the cages constituting each group were dispersed in batteries so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable. The position of the cage batteries in the room were changed weekly, following a rotation plan, to further minimise possible effects of spatial variations.
- Diet: Ad libitum. Main study animals did not have access to food overnight during the period of urine collection which preceded blood sampling procedures for haematology and blood chemistry investigations on the following day.
- Water: Ad libitum except during urine collection. Potable water from public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
- Acclimation period: Eight days before commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY: Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis are routinely provided by the water supplier. No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Monitored and maintained at 20-24 ºC. One one occasion during the treatment period (Day 21) the room temperature was outside the indicated range, but this was a minor deviation of short duration and was considered not to have influenced the health of the animals or the outcome of the study.
- Humidity (%): 40-70 %
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated. There was a minimum of 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light : 12 hours dark.
Route of administration:
oral: gavage
Details on route of administration:
Administration was via oral gavage using a suitably graded syringe and a rubber catheter inserted via the mouth. The oral gavage route of administration was chosen to simulate the conditions of potential human exposure.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS
- Starting with the lowest concentration, the required amount of test item was weighed into a suitable container. Vehicle was added to the mixture (to achieve approximately 50 % of the final volume) and stirred by hand using a spatula until the formulation was at the correct consistency and then magnetically stirred until uniformly mixed. The remaining vehicle was then added to make up to the required volume and then magnetically stirred until homogenous.
Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 2 and 200 mg/mL were analysed to assess the stability and homogeneity of the test item in the vehicle.
Adequate homogeneity and stability of the test item in the vehicle at these concentrations were achieved following refrigerated (2-8 °C) storage for up to 19 days and at ambient temperature (nominally 21 °C) for two days.
- Rate of preparation: The test doses were prepared weekly.
- A correction factor (including purity) of 1.429 was used when calculating quantities of test item used during dose preparation.
- Storage of formulation: Refrigerated (nominally 2 - 8 °C).

VEHICLE
- Justification for use and choice of vehicle: Not specified
- Concentration in vehicle: The test doses were prepared at nominal concentrations of 3.2, 8.0 and 20.0 mg/mL.
- Amount of vehicle: 5 mL/kg body weight.

STABILITY AND HOMOGENEITY
- Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 2 and 200 mg/mL were analyzed to assess the stability and homogeneity of the test item in the vehicle.
Adequate homogeneity and stability of the test item in the vehicle at these concentrations were achieved following refrigerated (2-8 °C) storage for up to 19 days and at ambient temperature (nominally 21 °C) for two 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 material.

TEST ITEM PREPARATION AND ANALYSIS
Control vehicle: Corn Oil
Propan-2-ol: HPLC grade
Water: Reverse osmosis
Formic Acid: Analytical reagent
Extract solvent and diluent: Propan-2-ol

PREPARATION OF CALIBRATION STANDARDS
A primary standard solution (1000 µg/mL) was prepared by dissolving an accurately weighed quantity (ca. 50 mg) of test material in Propan-2-ol (50 mL). Solutions for instrument calibration were prepared by appropriate dilution of the primary standard using Propan-2-ol and contained test material at nominal concentrations of 200 ng/mL, 400 ng/mL, 500 ng/mL, 600 ng/mL, 800 ng/mL and 1000 ng/mL. Calibration standards were matrix-matched by including 250 µL of the initial control vehicle extract.
Calibration solutions were injected onto the LC-MS/MS, at the beginning of each sample analysis sequence, using the conditions detailed in the chromatographic section.

PREPARATION OF TEST SAMPLES
A representative sample of test formulation (1 mL, accurately weighed) and dissolved using ultrasonic vibration in a suitable volume of Propan-2-ol. The extract was diluted using Propan-2-ol, to provide a solution containing test material at an expected concentration within the range 400 ng/mL to 800 ng/mL. Samples were matrix-matched by including an appropriate volume of the initial control vehicle extract in the final sample dilution where necessary.
The concentration of test material in the final solution was quantified by LC-MS/MS as detailed in the chromatographic section.

PREPARATION OF RECOVERY SAMPLES
Procedural recoveries were prepared by fortifying samples (1 mL) of control matrix (corn oil) with known amounts of test material. The prepared procedural recoveries were analyzed in accordance with the analytical procedure.

INSTRUMENTATION PARAMETERS
LC-MS/MS: Agilent 1100 Binary pump, Perkin Elmer PE200 Autosampler and Sciex API 3000 mass spectrometer or Ionics EP10+ mass spectrometer
Column: Agilent Poroshell SB C18, 2.7 µm, 100 x 4.6 mm
Column temperature: 50 °C
Sample temperature: Ambient
Mobile Phase A: Propan-2-ol/water/formic acid 50/50/0.1 v/v/v
Mobile Phase B: 0.1 % formic acid in Propan-2-ol

Linear Gradient
Time (minute) % A % B
0.0 60 40
4.0 0 100
7.0 0 100
7.1 60 40
10.0 60 40

Flow rate: 0.5 mL/minute
Rinse solvent/Needle wash: Propan-2-ol
Injection volume: 10 µL
Run time: 10 minutes
Approximate retention time: 2.75 minutes
MS Conditions:
Ionisation Mode: Turbo IonSpray Negative Ion Detection
Monitored Ions: m/z 283.4 239.3

CALCULATIONS
The peak area response for the test material in each calibration standard chromatogram was measured. Calibration curves were constructed by linear regression of calibration standard response versus calibration standard concentration. The area response of the peak observed at the characteristic retention time for test material in sample and procedural recovery chromatograms was measured. The response factor for the single bracketing standard was determined using the following equation:

- Response factor (RF) = Calibration standard peak response (Ac) / Concentration of calibration standard (ng/mL)

The concentration of test material was determined using the following equation:
- Analysed concentration (mg/mL) = (As/RF) x (V/W) x (D/100000)

Procedural recovery values were determined using the following equation:
- Procedural recovery (%) = (Analysed concentration (mg/mL)/ Fortified concentration (mg/mL)) x 100

Sample concentrations were corrected for procedural recoveries using the following equation:
- Corrected concentration, mg/mL = Analysed concentration, mg/mL x (100/R)

Where
Ac = Peak response for test material in single standard.
As = Peak response for test material in sample chromatogram.
RF = Mean response factor of appropriate set of bracketing standard
V = Dilution volume (mL)
W = Sample weight (g)
R = Appropriate procedural recovery value
D = Density of sample (g/mL)

VALIDATION OF THE ANALYTICAL PROCEDURE
The analytical procedure was validated by determining the following parameters: The specificity of the chromatographic analysis in control sample chromatograms.
The linearity of detector response over the calibration standard concentration range. The repeatability of the lowest and highest concentration calibration standards. The method accuracy and precision, by determining five procedural recoveries at nominal concentrations of 2 mg/mL and 200 mg/mL during the method validation.

Homogeneity and Stability in Corn Oil Formulations
The homogeneity and stability of the test material in corn oil formulations was assessed at nominal concentrations of 2 mg/mL and 200 mg/mL, during ambient and refrigerated storage. Freshly prepared specimen formulations (300 mL) were equally sub divided into four amber glass screw top bottles by Pharmacy personnel and submitted for analysis.

Ambient temperature storage (nominally +21 ºC)
On receipt, the contents of one bottle of each formulation were mixed by 20-fold inversion followed by magnetic stirring. After stirring for 20 minutes (representing 0 hour) and 4 hours, single samples (nominally 1 mL) were removed for analysis from the top, middle and bottom of the continuously stirred formulation.
The remainder of the bottle was stored at ambient temperature and after 2 days storage the contents were remixed and sampled as detailed above.

Refrigerated storage (nominally +4 ºC)
The remaining bottles were refrigerated on receipt and on Day 2 and Day 15; the appropriate remaining bottle was removed from storage and equilibrated to ambient temperature. The contents of the bottle were mixed by 20-fold inversion followed by magnetic stirring for 20 minutes and single samples (nominally 1 mL) were removed for analysis from the top, middle and bottom of the stirred formulation.
Bottle 4 was removed from storage and re-mixed on Day 19 due to an error with the Day 15 analysis. On Day 15 the appropriate bottles were not properly re-homogenized and the results of this analysis are not reported.

Concentration of Dose Formulations
For Week 1 and Week 4, freshly prepared test formulations were sampled (4 × 1 mL, accurately weighed) by Pharmacy personnel and submitted for analysis. Duplicate samples were analyzed in accordance with the analytical procedure, and the remaining samples were retained for contingency. Samples were disposed of once satisfactory results were achieved.

RESULTS
METHOD VALIDATION
The analytical procedure was successfully validated for the test material in corn oil with respect to the specificity of chromatographic analysis, the linearity of detector response, repeatability, method accuracy and precision.
The specificity of the LC-MS/MS assay was demonstrated by the absence of a peak with a response >20 % of the lowest standard at the characteristic retention time for the test material in the control sample chromatogram.
Linearity was confirmed over the nominal concentration range 200 ng/mL to 1000 ng/mL with a correlation coefficient r >0.995; the repeatability as <2 % for six replicate injections of standard solutions containing the test material at nominal concentrations of 200 ng/mL and 1000 ng/mL; Method accuracy and precision were confirmed:
A mean procedural recovery value of 104.8 % (CV=2.22 %, n=5) was obtained for 2 mg/mL and 108.9 % (CV=1.90 %, n=5) was obtained for 200 mg/mL.

HOMOGENEITY AND STABILITY OF DOSE FORMULATIONS
The homogeneity and stability of the test material in corn oil formulations was assessed with respect to the level of concentration at nominal concentrations of 2 mg/mL and 200 mg/mL Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for 4 hours, and on re-suspension following storage at ambient temperature for 2 days and refrigeration for up to 19 days. At each time-point, the mean analysed concentration for the three samples remained within 8 % of the initial time zero value and the coefficient of variation was less than 4 %.
The recoveries for the validation and whole trial were assessed and the overall mean of all the recoveries was used to set the acceptable limit for the stability assessment and routine occasions. This was due to the greater variability observed in the LC-MS/MS analysis. The acceptable range was found to be 98.1 % to 113.1 %. Recovery results during the trial remained within the acceptable range of the mean recovery. The trial samples were corrected for the mean of each level, each occasion recoveries.

CONCENTRATION OF DOSE FORMULATIONS
For Week 1 the mean concentrations of the test material were within applied limits +10/-15 % of nominal, confirming the accuracy of formulation.
For Week 4 the mean concentrations of the test material were not within applied limits +10/-15 % of nominal.
An investigation into the Week 4 results was conducted. The analysis results were confirmed by re-dilution in duplicate and the recovery results were found to be acceptable with the exception of one recovery at 92.3 % for 8 mg/mL level (this was rejected in line with the SOP). Consequently analytical error was eliminated as a possible cause of the low mean achieved concentration results. The formulation documentation showed that the correct weight of test item and volume of vehicle were used to prepare the Week 4 formulations. The test item has a high viscosity and on viewing a preparation for another related study it was noted that a step in the formulation preparation method could potentially cause issues if the test item is not mixed properly with corn oil. Losses would occur on this initial mixing step where test item is mixed with approximately half of the required volume of vehicle in order that it is moveable enough to magnetically stir.
It was not possible to confirm that a mixing error was the cause of the low results, however it is deemed that the analytical results are valid and robust and that an error at the time of formulation preparation was the most likely cause. It is believed that the Week 2 and Week 3 formulations which were not sampled were prepared correctly. The formulation preparation documentation shows that the correct weight of test item and the correct volume of vehicle were used. In addition, formulations for Weeks 2 and 3 were prepared by technicians who were involved in the preparation of the Week 1 and stability trial preparations (where achieved concentrations were found to be within acceptable limits in both cases).
Consequently, it is considered that any potential of a formulation preparation error would have been confined to Week 4 only. The formulation preparation method has been updated to include further information about the mixing step and observations to avoid further errors in this step of the preparation.

CONCLUSION
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, repeatability, method accuracy and precision.
The homogeneity and stability was confirmed for the test material in corn oil formulations at nominal concentrations of 2 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 4 hours, ambient temperature storage for 2 days and refrigerated storage for up to 19 days.
The mean concentrations of the test material in test formulations analysed for Week 1 of the study were within +10 %/-15 % of nominal concentrations, confirming accurate formulation. The Week 4 results were low (-18 % to -25 %) and were investigated.
Duration of treatment / exposure:
28 days (four weeks)
Frequency of treatment:
Once daily at approximately the same time each day.
Dose / conc.:
16 mg/kg bw/day (nominal)
Dose / conc.:
40 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 animals per sex per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels selected for this study (16, 40 and 100 mg/kg/day) were selected based on the results of a 14-day dose-range finding study.
In that study, doses of 300 and 1000 mg/kg/day were not tolerated and resulted in a range of post-dose observation signs which predominantly included, but were not limited to, decreased activity and piloerection with some animals (predominantly females receiving 1000 mg/kg/day) also being abnormally cold to touch and having hunched posture. The extent and nature of these signs necessitated the premature termination of both sexes receiving 1000 mg/kg/day and females receiving 300 mg/kg/day during the early stages of the study; animals were humanly killed before or after dosing on Day 2 or after dosing on Day 3 at the 1000 or 300 mg/kg/day dose levels, respectively. Predominant findings at necropsy at 300 and/or 1000 mg/kg/day included abnormalities of the gastro-intestinal tract, comprising of firm content in the caecum and ileum, pale content in the jejunum and a range of findings in the stomach including, but not limited to, distended appearance (at 1000 mg/kg/day only), irregular surface or thickening of the non-glandular region and depressions in the glandular mucosa. At 100 mg/kg/day, possible test substance-related post-dose signs were limited to transient incidences of piloerection seen on Day 4, a few transient incidences of post-dose chin rubbing (where animals were seen to push their muzzles through the cage bedding material; a finding possibly associated with the oral gavage route of administration) on Day 13 and an isolated/transient incidence of noisy breathing (rales) in one male on Day 13 (also a finding that may had been related to the oral gavage route of administration). Macroscopic gastrointestinal findings at 100 mg/kg/day were confined to a single incidence of a thickened non-glandular region of the stomach in one female.

Based on this information, it was considered appropriate to investigate a high dose level of 100 mg/kg/day in this study. All dose levels were expected to be tolerated over a 4-week administration period. The intermediate and low dose levels were selected to assess any dose-responsiveness of any test substance-related finding following a longer (i.e. four week) treatment period.

- Rationale for animal assignment: Randomly allocated on arrival. Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately.

- Post-exposure recovery period: Animals dosed with the vehicle control and animals dosed with 100 mg/kg/day were subjected to a 2-week recovery period. Cessation of treatment for the recovery phase animals started on the day of the necropsy of animals allocated to the main study. Serial observations were recorded at appropriate intervals.
Positive control:
None
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
Signs are considered in two parts: Detailed observations recorded at times in relation to dose administration, classified as ‘signs associated with dosing’ and extended changes in condition, classified as ‘detailed physical examination and arena observations’. Detailed physical examination and arena observations are presented for each animal, providing detail of type of sign, week of occurrence and information on the duration of the sign applicable.

- Clinical and Behavioral Observations: 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 and recovery periods, observations of the animals and their cages were recorded at least once per day.

- Signs Associated with Dosing: Observed daily during the treatment period, detailed observations were recorded at the following times in relation to dose administration:
During Week 1 of treatment there was a pre-dose observation as well as observations at the end of dosing each group, one to two hours after completion of dosing of all groups and as late as possible in the working day.
During Week 2 of treatment to termination here was a pre-dose observation as well as observation one to two hours after completion of dosing of all groups.

- Detailed Physical axamination and arena Observations: 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.
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
- 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. The last scheduled body weight was recorded on Day 28 for the main study animals (prior to overnight deprivation of food and water for clinical pathology investigations) and Day 14 of recovery for the recovery phase animals. Group mean weight changes were calculated from the weight changes of individual animals.

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. Food remaining for the last week of the treatment and recovery periods was recorded on Day 28 (i.e. prior to overnight deprivation of food for clinical pathology investigations) and Day 14, respectively. Weekly group mean food consumptions and standard deviations were derived from unrounded cage values. Overall mean food consumption values were calculated from the weekly group mean values.

WATER CONSUMPTION: Yes
Fluid intake was assessed by daily visual observation. No test item-related effects were observed and, consequently, quantitative measurements were not performed.

HAEMATOLOGY: Yes
Blood samples were collected on day 29 from all main study animals and on day 15 of recovery from all the recovery animals.
Samples taken from the main study animals on Day 29 were collected after overnight withdrawal of food and prior to dosing. Sampling was performed on the day after overnight collection of urine so the animals were 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. Animals sampled on Day 15 of recovery had access to food and water prior to sampling. Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics:
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), large unstained cells) and platelet count (Plt).
‡ = Recovery phase animals were only examined for these parameters

Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate.

Additional blood samples (nominally 0.5 mL) were taken from Main study animals (on Day 29) into tubes containing citrate anticoagulant and examined using an ACL series analyzer and appropriate reagent in respect of:
Prothrombin time (PT) - using IL PT Fibrinogen reagent.
Activated partial thromboplastin time (APTT) - using IL APTT reagent.

CLINICAL CHEMISTRY: Yes
- Blood samples were collected on day 29 from all main study animals. Blood sampling was performed on the day after overnight collection of urine. Animals, were, therefore, 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. Animals were held under light general anesthesia induced by isoflurane. 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 Analyzer in respect of:
Alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (gGT), total bilirubin (Bili), total bile acids (BiAc), urea, creatinine (Creat), glucose (Gluc), total cholesterol (Chol), triglycerides (Trig), sodium (Na), potassium (K), chloride (Cl), inorganic phosphorus (Phos), total protein (Total Prot) and albumin (Alb).
Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.

Albumin to globulin ratio (A/G Ratio) was calculated as:

A/G Ration = Albumin concentration / (total protein - albumin concentration)

URINALYSIS: Yes
Animals were placed in an individual metabolism cage, without food or water. Urine samples were collected overnight on day 29 from all main study animals.

The individual samples were examined for the following characteristics:
- Using manual methods: Clarity and colour - by visual assessment, volume (Vol) - using a measuring cylinder, pH - using a pH meter and specific gravity (SG) - by direct refractometry using a SG meter.
- Using Multistix reagent strips interpreted using the Clinitek®500 instrument: Ketones (Keto), bile pigments (Bili) and blood pigments (UBld).
- Using a Roche P Modular Analyzer: Protein (T-Prot), creatinine (T-Creat), glucose (T-Gluc), sodium (T-Na), potassium (T-K) and chloride (T-Cl).

A microscopic examination of the urine sediment was performed. An aliquot of the urine sample was centrifuged, stained with Kova stain and the resulting deposit spread on a microscope slide. The number of elements seen in nine high or low power fields (HPF or LPF) was recorded in the raw data and entered onto the database and the number seen /HPF or /LPF was derived from these data as described:
Epithelial cells (Epi), leucocytes (WBC), erythrocytes (RBC), casts and other abnormal components (A).

The slide was also examined for abnormalities in spermatozoa and crystals.

The appearance of urine was described in the appendix using the following abbreviations:
MY Clear medium yellow appearance
MO Clear medium orange appearance
DY Clear dark yellow appearance
CMY Cloudy medium yellow appearance

Multistix are diagnostic reagents and were used as qualitative indicators of analyte concentration. Results are reported according to the following convention:
NEG negative
TRA trace levels of analyte detected
1+ small amount of analyte detected
2+ moderate amount of analyte detected
3+ large amount of analyte detected
4+ very large amount of analyte detected

Group means and standard deviations are presented for volume, pH, specific gravity, protein, creatinine, glucose and electrolytes only.

NEUROBEHAVIOURAL EXAMINATION:
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. 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
A blunt probe was brought towards the animal’s head until it was close to the animal’s nose (but not touching the whiskers). The animal’s reaction was recorded as:
1 No reaction or ignores probe/walks past probe
2 Normal awareness and reaction e.g. approaches and/or sniffs probe
3 Active avoidance, abnormally fearful or aggressive reaction

- Pinna reflex
The inside of one ear was touched lightly with a nylon filament and the reaction recorded as:
1 No response
2 Normal response e.g. ear twitches/flattens or animal shakes its head
3 Abnormally fearful or aggressive response

- Auditory startle reflex
The animal’s response to a sudden sharp noise was assessed and scored as:
1 No response
2 Weak response e.g. ear twitch only
3 Normal response e.g. obvious flinch or startle
4 Exaggerated response e.g. all feet off floor

- Tail pinch response
The animal’s tail was pinched sharply with forceps approximately one third from the tip and the response graded as:
1 No response
2 Weak response e.g. turns around slowly or weak vocalization without moving away
3 Normal response e.g. jumps forward or turns around sharply, usually with vocalization
4 Exaggerated response e.g. excessive vocalization, body movement or aggression

- Grip strength
Forelimb and hindlimb grip strength was measured using Mecmesin Basic Force Gauges. Three trials were performed. At any point during the observations, additional comments were made as free text where considered appropriate.

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. 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.
Sacrifice and pathology:
METHOD OF KILL
Carbon dioxide asphyxiation with subsequent exsanguination.

NECROPSY
All main study and recovery 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. The retained tissues were checked before disposal of the carcass. The organs weighed, tissue samples fixed and sections examined microscopically are detailed in Table 1.

ORGAN WEIGHTS
For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for main study and recovery animals killed at scheduled intervals.
Organ weights were presented both as absolute/unadjusted and adjusted for terminal body weight, using the weight recorded on the day of necropsy.

FIXATION
Tissues were routinely preserved in 10 % Neutral Buffered Formalin with the exception of testes which were fixated in modified Davidson’s fluid and eyes which were fixated 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: Main study animals of Groups 1 and 4.
- Abnormalities only: All main study animals of Groups 2 and 3 and all recovery phase animals.
- Routine staining: Sections were stained with hematoxylin and eosin.

LIGHT MICROSCOPY
Tissues preserved for examination were examined as follows:
- All tissues specified in Table 1 for all main study animals of Groups 1 and 4 at terminal sacrifice
- All tissues with abnormalities from the main study animals of Groups 2 and 3 at terminal sacrifice.
- All tissues with abnormalities from the recovery phase animals at recovery sacrifice.

Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.
Statistics:
See below.
Clinical signs:
no effects observed
Description (incidence and severity):
The general appearance and behavior of the animals was unaffected by treatment.
Mortality:
no mortality observed
Description (incidence):
There were no deaths observed.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no effect of treatment on body weight.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was unaffected by treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
A visual assessment of water intake did not reveal any clear or consistent effects of treatment. Some inter-group differences were apparent (males receiving 100 mg/kg/day appeared to consume more water than controls on Day 21 of the treatment phase and animals which previously received 100 mg/kg/day appeared to consume more water than the controls on Day 12 (males only) and 14 (both sexes) of the recovery phase) but these were isolated findings and therefore considered to be unrelated to treatment; especially given that no clear or persistent effect was evident during the treatment period.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clear test item-related findings.
Some inter-group differences, predominantly seen in red or white blood cell parameters, from the controls attained statistical significance at the high dose level (100 mg/kg/day) but in the majority of cases, the differences from controls were relatively minor and were not consistent between the sexes and, as such, had an uncertain relationship to treatment.

Differences in red blood cell parameters seen at the end of the treatment period included slightly low hematocrit and red cell distribution width in males and slightly low hemoglobin concentration in females. No similar trends were seen following two weeks cessation of dosing with the exception of hematocrit which remained slightly low, but not statistically significantly different compared to controls, in males which had previously received 100 mg/kg/day.

Differences in white blood cell parameters seen at the end of the treatment period included slightly high monocyte count in males and slightly low neutrophil and total white blood cell counts in females. Following two weeks cessation of dosing, mean monocyte counts remained slightly high and mean neutrophil counts remained slightly low in males and females, respectively, which previously received 100 mg/kg/day but the differences did not attain statistical significance when compared with the controls.

Other statistically significant inter-group differences seen at the end of the treatment period were confined to slightly low platelet counts in females which received 100 mg/kg/day and a slight decrease in prothrombin time without dose-relationship in all treated groups of females. Neither finding was evident at the end of the 2-week recovery period.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The biochemical investigation on Day 28 did not reveal any test item-related changes.
All inter-group differences from controls, including those which attained statistical significance, were generally minor, confined to one sex only or lacked any clear dose-response and were, therefore, considered to be due to normal biological variation. Such differences included, but were not limited to, the slightly high aspartate amino-transferase activity in males which received 100 mg/kg/day (which was attributable to slightly high values in three animals, Nos. 6, 9 and 10, and where no similar trend was evident in females), the slight disturbances in chloride concentrations seen in animals which received 40 or 100 mg/kg/day (where the differences showed no consistency in the direction of change from controls between the sexes) and the marginally low glucose concentration seen in all treated groups of males (which lacked dose-response and where no similar trend was evident in females).
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalysis performed at the end of the treatment period did not reveal any test item-related findings.
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Analysis of organ weights after 4 weeks of treatment revealed marginally low mean absolute and body weight-adjusted spleen weights (without dose-response) for males which received 40 or 100 mg/kg/day. No similar trend was evident at the end of the two-week recovery period where mean absolute and body weight-adjusted spleen weights were similar to those of the controls for males which had previously received 100 mg/kg/day.

All other inter-group differences from controls, including those which attained statistical significance, were generally minor, confined to one sex only or lacked any clear dose-response and were, therefore, considered to be due to normal biological variation.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The macroscopic examination performed after 4 weeks of treatment or 2 weeks of recovery did not reveal any test item-related lesions.
The incidence and distribution of all findings were consistent with the common background of macroscopic changes seen at these laboratories.
Neuropathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
SENSORY REACTIVITY AND GRIP STRENGTH
Sensory activity and grip strength were unaffected by treatment.

MOTOR ACTIVITY
Motor activity scores were not affected by treatment.
The mean total low and high beam break scores (cage floor and rearing activity, respectively) were marginally low for all treated groups of males, when compared with the controls, but there was no dose response, the differences did not attain statistical significance and no similar trend was observed in females. Furthermore, the group mean values for total low and high beam break scores for the treated groups of males were within the recent historical control data range (n=3 studies). In contrast, the group mean total low beam break score for the control males was above the maximum range of historical control data. Consequently, these were considered incidental differences.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
- Animals killed after 4 Weeks of treatment
No changes related to treatment with test material were seen. Minimal hyaline droplet accumulation was seen in three males treated at 100 mg/kg/day but this finding had a doubtful relationship to treatment since a single incidence, at the same severity, was also evident in one control male. This finding is commonly seen in male rodents.
All other microscopic findings were also considered incidental and unrelated to the test item.

- Animals killed after 2 Weeks of recovery
No changes related to treatment with test material were seen in animals previously treated for 4 weeks followed by a 2 week recovery period.
All microscopic findings were considered incidental and unrelated to the test item.
Histopathological findings: neoplastic:
no effects observed
Details on results:
The oral administration of test material to Han Wistar (RccHan™;WIST) rats for 4 weeks at dosages up to 100 mg/kg/day was well tolerated with no adverse effect of treatment.
The achieved concentration results of formulations that were prepared for administration during the final week (Week 4) of treatment were lower than expected, with mean concentrations being -18.4, -23.6 and -25.5 % of nominal concentration for Groups 2 (3.2 mg/mL), 3 (8 mg/mL) and 4 (20 mg/mL), respectively. Whilst it was not possible to confirm the exact cause of these low results, an error at the time of formulation preparation (possibly due to inadequate mixing procedures) was the most likely cause given that there was no evidence of analytical failure. Overall, it is considered that all treated groups of animals would have been adequately exposed to the test material formulations in this study and that receiving slightly lower than intended dose levels during the final week of treatment did not have any significant impact on the overall interpretation of study findings.

Some inter-group differences in hematology parameters were evident at the end of the treatment period in animals which received 100 mg/kg/day but the differences from controls were relatively minor and were not consistent between the sexes and, as such, had an uncertain relationship to treatment. The slightly low hematocrit, red cell distribution width and/or hemoglobin concentrations were minor changes that were not seen in association with any other effects in red blood cell parameters (total red blood cell count, reticulocyte count and mean cell volume were all unaffected) and were either not evident at the end of the recovery period or showed no progression of apparent change. There was a marginal reduction in spleen weight at the end of the treatment period in males which received 40 or 100 mg/kg/day but the cause of this finding was not established (there was no microscopic correlate from evaluation of the spleen from males which received 100 mg/kg/day) and there was evidence of recovery at the end of the 2-week off-dose period. Consequently, none of these changes was considered adverse.

Similarly, the apparent increase in monocyte count and reduction in neutrophil and total white blood cell counts seen at the end of the treatment period had an uncertain relationship to the test item, given the absence of any consistency between the sexes, but were non-adverse changes due to their minor nature and the absence of any test item-related histopathological change in a range of lymphoid tissues (bone marrow, thymus, lymph nodes and spleen) which were examined from animals given 100 mg/kg/day.

The cause of the apparent reduction in platelet count in females at 100 mg/kg/day and decrease in prothrombin time at all dose levels in females was not established but these were considered non-adverse findings (given the relatively small magnitude of change and absence of any other study findings indicative of an adverse effect on clotting function) which showed complete recovery.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no treatment-related effects at the highest dose tested.
Key result
Critical effects observed:
no

Formulation Analysis

The mean concentrations of the test material in the formulations prepared for Week 1 were between -6.8 % and -12.2 % of nominal concentrations for all groups. These were within the applied limits of -15 % / +10 % of the nominal concentrations, demonstrating accurate formulation.

The nominal concentrations for Group 2 (3.2 mg/mL), Group 3 (8 mg/mL) and Group 4 (20 mg/mL) in Week 4 were -18.4 %, -23.6 % and -25.5 % of the nominal concentrations, respectively. The exact cause of these low concentrations could not be established but since no analytical errors were evident, it is suspected that there may have been a formulation error at this occasion.

It is considered that exposure of the test substance to animals assigned to the treated groups would have been close to nominal in terms of expected dose levels (on a mg/kg/day basis) for three out of the four weeks of treatment. Overall, it is considered that all treated groups of animals would have been adequately exposed to test material formulations in this study and that receiving lower than intended dose levels during the final week of treatment did not have any significant impact on the overall interpretation of the findings in this study and/or the establishment of the No-Observed-Adverse-Effect-Level (NOAEL).

Table 2: Results of formulation analysis

Week of Formulation

Group

Nominal

Analysed concentration

RME (%)

CV (%)

inclusion

(mg/mL)

(mg/mL)

Analysis 1

Analysis 2

Mean

Week 1

1

0

ND

ND

-

-

-

2

3.2

2.83

2.79

2.81

-12.2

0.92

3

8

7.59

7.32

7.46

-6.8

2.53

4

20

18

17.6

17.8

-11.0

1.51

 

 

 

 

 

 

 

 

Week 4

1

0

ND

ND

-

-

-

2

3.2

2.61

2.61

2.61

-18.4

0.15

3

8

6.25

5.97

6.11

-23.6

3.26

4

20

13.1

16.7

14.9

-25.5

17.32

ND Not detected

Conclusions:
Under the conditions of the study, the No Observed Adverse Effect Level (NOAEL) was considered to be 100 mg/kg/day.
Executive summary:

The potential for the test material to cause repeated dose toxicity was investigated in a GLP study conducted in accordance to the standardised guidelines OECD 407, EU method B7, OPPTS 870.3050 and Japanese guidelines Yakushokuhatsu No. 0331. 7, Seikyoku No. 5 and Kanpokihatsu No.110331009.

The systemic toxic potential of the test material was investigated in a 4-week oral gavage study in the Han Wistar (RccHan™;WIST) rat. Recovery from any effects was evaluated during a 2-week recovery period.

Main study animals received the vehicle (corn oil) or the test material (16, 40 or 100 mg/kg/day) by oral gavage for four weeks. Recovery animals were similarly treated for four weeks followed by a two-week off dose period.

During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood), blood chemistry, urinalysis, organ weight, macropathology and histopathology investigations were undertaken.

The achieved concentration results of formulations that were prepared for administration during the final week (Week 4) of treatment were lower than expected. Whilst it was not possible to confirm the exact cause of these low results, an error at the time of formulation preparation (possibly due to inadequate mixing procedures) was the most likely cause given that there was no evidence of analytical failure. Overall, it is considered that all treated groups of animals would have been adequately exposed to the test material formulations in this study and that receiving slightly lower than intended dose levels during the final week of treatment did not have any significant impact on the overall interpretation of study findings.

There were no deaths observed throughout the study.

The general appearance and behavior, sensory activity, grip strength and motor activity of the animals were not affected by treatment.

There was no effect of treatment on body weight gain or food consumption and a visual assessment of water intake did not reveal any suspicion of any treatment-related effect.

The haematological investigation at the end of the treatment period revealed a few findings of uncertain relationship to treatment at 100 mg/kg/day (slightly low haematocrit and red cell distribution width in males, slightly low haemoglobin concentration in females, slightly high monocyte count in males and slightly low neutrophil and total white blood cell counts in females) but none was associated with any test item-related histopathological change. Some differences were still apparent at the end of the 2-week recovery period (haematocrit remained slightly low in males and monocyte counts remained slightly high and neutrophil counts remained slightly low in males and females, respectively) but none of these differences attained statistical significance.

Slightly low platelet count occurred in females at 100 mg/kg/day and a slight decrease in prothrombin time occurred in all treated groups of females (without dose-relationship) at the end of the treatment period but neither finding was evident at the end of the 2-week recovery period.

The biochemical examination of the blood and urinalysis performed at the end of the treatment period did not reveal any test item-related findings.

Marginally low spleen weights were evident at the end of the treatment period in males at 40 or 100 mg/kg/day but there was no associated histopathological correlate and the finding showed complete recovery.

None of the above differences from control was considered adverse. There were no treatment related macroscopic or microscopic findings.

It is concluded that oral administration of the test material to Han Wistar (RccHan™;WIST) for 4 weeks at dosages up to 100 mg/kg/day was well tolerated. There were no target organs identified at histopathological examination.

Under the conditions of the study, the No Observed Adverse Effect Level (NOAEL) was considered to be 100 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

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

14 Day Range-Finding Study: Oral Route

The test material was administered orally by gavage to Han Wistar (RccHanTM;WIST) rats for 14 days at dose levels of 30, 100, 300 and 1000 mg/kg/day in order to assess the systemic toxic potential. During the study, clinical condition, body weight, food consumption, visual water consumption assessment, organ weight and macropathology investigations were undertaken.

For the animals dosed at 1000 mg/kg/day, signs after dosing included decreased activity, piloerection, partially closed eyes, a thin build, a hunched posture and elevated gait. All animals were killed prematurely on Day 2 due to the severity of the signs observed.

Necropsy revealed incidences of distended stomach, dark areas and/or irregular surface on the non-glandular mucosa and depressions on the glandular mucosa. Other changes included dark areas in the medulla of the kidneys (females only) and some other abnormalities of the gastrointestinal tract including incidences of firm contents in the ileum and caecum. Low spleen weights were noted for two females. For the animals dosed at 300 mg/kg/day, signs after dosing for the females included decreased activity, piloerection, abnormally cold to touch and abnormal vocalisation and they were subsequently killed prematurely on day 3 due to the severity of the signs observed. For the males, decreased activity, piloerection and chin rubbing after dosing were observed from day 3, although the condition of the animals did not necessitate premature sacrifice. All males lost bodyweight over the first three days of treatment although all started to gain weight from day 4. Food consumption for males was low throughout the treatment period. There was no clear effect of treatment on organ weights. Necropsy of the females killed prematurely revealed an irregular surface on the non-glandular mucosa and depressions on the glandular mucosa of the stomach. Other changes included dark areas in the medulla of the kidneys. For the males killed on day 15, necropsy revealed a thickened non-glandular mucosa of the stomach in all three animals and a dilated pelvis of the kidneys in two of them. For the animals dosed at 100 mg/kg/day, the appearance and behaviour of the animals were unaffected by treatment and there were no deaths. There were no effects on bodyweight gain or food consumption. There was no clear effect of treatment on organ weights. Necropsy revealed a dilated pelvis of the kidneys in three animals (one male and two females) and thickened non-glandular mucosa of the stomach in one female. For the animals dosed at 30 mg/kg/day The appearance and behaviour of the animals were unaffected by treatment and there were no deaths. There were no effects on bodyweight gain or food consumption. There was no clear effect of treatment on organ weights and no findings at necropsy.

It is therefore concluded that under the conditions of the study, administration of the test material to Han Wistar rats was well tolerated at doses up to 100 mg/kg/day. Treatment at 300 and 1000 mg/kg/day resulted in a range of signs which necessitated the premature termination of both sexes at 1000 mg/kg/day and females at 300 mg/kg/day.

 

28 Day Repeated Dose Toxicity: Oral Route

The potential for the test material to cause repeated dose toxicity was investigated in a GLP study conducted in accordance to the standardised guidelines OECD 407, EU method B7, OPPTS 870.3050. and Japanese guidelines YAKUSHOKUHATSU No. 0331. 7, SEIKYOKU No. 5 and KANPOKIHATSU No.110331009.

The systemic toxic potential of the test material was investigated in a 4-week oral gavage study in the Han Wistar (RccHan™;WIST) rat. Recovery from any effects was evaluated during a 2-week recovery period. Main study animals received the vehicle (corn oil) or the test material) by oral gavage for four weeks. Recovery animals were similarly treated for four weeks followed by a two-week off dose period. During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, haematology (peripheral blood), blood chemistry, urinalysis, organ weight, macropathology and histopathology investigations were undertaken.

The achieved concentration results of formulations that were prepared for administration during the final week (Week 4) of treatment were lower than expected. Whilst it was not possible to confirm the exact cause of these low results, an error at the time of formulation preparation (possibly due to inadequate mixing procedures) was the most likely cause given that there was no evidence of analytical failure. Overall, it is considered that all treated groups of animals would have been adequately exposed to the test material formulations in this study and that receiving slightly lower than intended dose levels during the final week of treatment did not have any significant impact on the overall interpretation of study findings.

There were no deaths observed throughout the study. The general appearance and behaviour, sensory activity, grip strength and motor activity of the animals were not affected by treatment. There was no effect of treatment on body weight gain or food consumption and a visual assessment of water intake did not reveal any suspicion of any treatment-related effect. The haematological investigation at the end of the treatment period revealed a few findings of uncertain relationship to treatment at 100 mg/kg/day (slightly low haematocrit and red cell distribution width in males, slightly low haemoglobin concentration in females, slightly high monocyte count in males and slightly low neutrophil and total white blood cell counts in females) but none was associated with any test item-related histopathological change. Some differences were still apparent at the end of the 2-week recovery period (haematocrit remained slightly low in males and monocyte counts remained slightly high and neutrophil counts remained slightly low in males and females, respectively) but none of these differences attained statistical significance. Slightly low platelet count occurred in females at 100 mg/kg/day and a slight decrease in prothrombin time occurred in all treated groups of females (without dose-relationship) at the end of the treatment period but neither finding was evident at the end of the 2-week recovery period. The biochemical examination of the blood and urinalysis performed at the end of the treatment period did not reveal any test item-related findings. Marginally low spleen weights were evident at the end of the treatment period in males at 40 or 100 mg/kg/day but there was no associated histopathological correlate and the finding showed complete recovery. None of the above differences from control was considered adverse. There were no treatment related macroscopic or microscopic findings. It is concluded that oral administration of the test material to Han Wistar (RccHan™;WIST) for 4 weeks at dosages up to 100 mg/kg/day was well tolerated. There were no target organs identified at histopathological examination.

Under the conditions of the study, the No Observed Adverse Effect Level (NOAEL) was considered to be 100 mg/kg/day.

Justification for waiving the 90 day repeated dose toxicity study - see the attachment in the 90 day repeated dose toxicity waiver Endpoint Study Record ("Attached Background Material" section)

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to repeated dose toxicity via the oral route.