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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:
22 April 2016 to 2 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

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: OPPTS 870.3050, Repeated Dose 28-Day Oral Toxicity Study in Rodents, July 2000.
Deviations:
no
Qualifier:
according to
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. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Test material form:
liquid: viscous
Details on test material:
Appearance: Brown viscous liquid
Storage: Room temperature in the dark under nitrogen

Test animals

Species:
rat
Strain:
Wistar
Remarks:
RccHan™;WIST rat.
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 and environmental conditions:
TEST ANIMALS
- Age at study initiation: Main study males and recovery phase animals were 38 to 44 days old. Main study females were 41 to 47 days old.
- Weight at study initiation: 116 to 160 g for males and 106 to 144 g for females. On Day 1 (before dosing) variations in body weight of the animals were checked to ensure that they did not exceed ± 20 % of the mean for the appropriate sex. Two females were rejected during the acclimatization period due to body weight range extremes and were replaced with spare animals of suitable weight from the same batch. In addition, a further eight females were swapped within their previously allocated cages/groups to balance group means.
- Fasting period before study: Not specified.
- Housing: Five animals of the same sex were housed in cages of polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals.
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 positions of the cage batteries in the room were changed weekly, following a rotation plan, to further minimize possible effects of spatial variations.
- Diet: Ad libitum.
- Water: Ad libitum.
- Acclimation period: Main study males and recovery phase animals were acclimatized for nine days before commencement of treatment. Main study females were acclimatised for 12 days before commencement of treatment. Spare animals were removed from the study room after treatment commenced.

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): 20 - 24 °C
- Humidity (%): 40 - 70 %.
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated (15 changes per hour).
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light : 12 hours dark.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route of administration was chosen as it was anticipated route of potential human exposure. The test item was administered by gavage.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- Correction factor: A correction factor (including purity) of 1.205 was used when calculating quantities of test item used during dose preparation.
- Method of preparation: A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test item. Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure. For Groups 2, 3 and 4, formulations were stirred for a minimum of thirty minutes immediately prior to commencement of dosing.
Treatment was at a constant dose in mg/kg/day.
Individual dose volumes were calculated from the most recently recorded scheduled body weight and were administered orally by gavage using a suitable graduated syringe and a rubber catheter inserted via the mouth.
- Rate of preparation (frequency): Weekly and in advance of the first day of dosing.
- Storage of formulation: Refrigerated (nominally 2-8 °C).

VEHICLE
- Justification for use and choice of vehicle: The test material was stable in corn oil formulations.
- Concentration in vehicle: At nominal concentrations of 20, 60 and 200 mg/mL and formulated concentration of 24.1, 72.3 and 240.6 mg/mL.
- Amount of vehicle: 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- 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 analysed to assess the stability and homogeneity of the test item in the liquid matrix.
It was established that adequate homogeneity and stability of the test substance in the vehicle at nominal concentrations of 2 mg/mL, 10 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 4 hours, at ambient temperature storage for 2 days (10 mg/mL and 200 mg/mL) and under refrigerated storage for up to 15 days (10 mg/mL and 200 mg/mL) and 22 days (2 mg/mL).

- Achieved concentration
Samples of each formulation prepared for administration in Weeks 1 and 4 of treatment were analysed for achieved concentration of the test item.

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 the 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 (1 mL vehicle diluted to 100 mL with propan-2-ol). 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) was 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 the test material at an expected concentration of 500 ng/mL. Samples were matrix matched by including an appropriate volume of the initial control vehicle extract in the final sample dilution where necessary to match the standards. The concentration of the 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 analysed in accordance with the analytical procedure.

INSTRUMENTATION PARAMETERS
Liquid chromatograph with mass spectrometry detection LC-MS/MS: Agilent 1100 Binary pump, Perkin Elmer PE200 Autosampler and Sciex API 300 (or API 365) mass spectrometer or Ionics EP10+ mass spectrometer.
Column: Varian Polaris C18-Ether, 3.0 µm, 50 × 2.1 mm
Column temperature: 50 °C
Sample temperature: Ambient
Mobile Phase A: Propan-2-ol/water/formic acid 25/75/0.1 v/v/v
Mobile Phase B: 0.1 % formic acid in propan-2-ol

Linear Gradient
Time (minute) %A %B
0.0 100 0
0.5 100 0
2.5 10 90
7.0 10 90
7.1 100 0
10.0 100 0

- Flow rate: 0.4 mL/minute
- Rinse solvent/needle wash: Propan-2-ol
- Injection volume: 10 µL
- Run time: 10 minutes
- Approximate retention time: 3.0 minutes

MS CONDITIONS
- Ionization Mode: Turbo IonSpray Positive Ion Detection
- Source Temperature: +500 °C
- Collision gas: Nitrogen
- Collision energy: 50 eV
- Dwell Time: 950 ms
- Pause Time: 5 ms
- Monitored Ions: m/z 855.1 ¿ 462.5

CALCULATIONS
The peak area response for the test material in each standard chromatogram was measured. Curves to assess linearity were constructed by linear regression (1/x weighting) of linearity standard response versus linearity standard concentration. The area response of the peak observed at the characteristic retention time for the 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 the test material was determined using the following equation:

Analysed concentration (mg/mL) = (As / RF) x (V / W) x (D / 1 000 000)

Procedural recovery values were determined using the following equation:

Procedural recovery = (Analysed concentration (mg/mL) / Fortified concentration (mg/mL)) x 100

The sample concentrations were corrected for the appropriate procedural recovery value using the following equation:

Corrected concentration, mg/mL = Analysed concentration, mg/mL x (100 / R)

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

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 limit of quantification.
- The linearity of detector response over the standard concentration range.
- The repeatability of the lowest and highest concentration linearity standards.
- The method accuracy and precision, by determining a minimum of five procedural recoveries at nominal concentrations of 2 mg/mL, 10 mg mL and 200 mg/mL.
- Attempts were made to prove extract stability for 7 days and standard stability for 8 days.

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, 10 mg/mL and 200 mg/mL, during ambient temperature and refrigerated storage. Freshly prepared specimen formulations (300 mL) were equally sub divided into four amber glass screw-top bottles (three bottles for Trial 3) and submitted for analysis.

AMBIENT TEMPERATURE STORAGE (15 - 25 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 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 (2 – 8 °C)
The remaining bottles were refrigerated on receipt and on Day 2, Day 8 (Trials 2 and 3), Day 15 and Day 22 (Trial 1); the appropriate 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 a minimum of 20 minutes and single samples (nominally 1 mL) were removed for analysis from the top, middle and bottom of the continuously stirred formulation.

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 analysed in accordance with the analytical procedure, and the remaining samples were retained for contingency. Samples were disposed of once analysis was complete.

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. Results are summarized below:
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 1 000 ng/mL with a correlation coefficient r>0.99;
The repeatability was <4 % for six replicate injections of standard solutions containing test material at nominal concentrations of 200 ng/mL and 1 000 ng/mL.

The original results for method accuracy and precision gave a mean procedural recovery value of 97.6 % (CV=1.69, n=5) for 2 mg/mL and 101.2 % (CV=0.95, n=5) for 200 mg/mL. During Trial 1 the 200 mg/mL recovery samples were not consistent with the original validation. It was decided to repeat the method accuracy and precision experiment. The method was also validated at 10 mg/mL for Trial 2. For the re-validation, method accuracy and precision were confirmed. A mean procedural recovery value of 101.7 % (CV=3.56, n=5) was obtained for 2 mg/mL, 98.7 % (CV=2.18, n=5) was obtained for 10 mg/mL and 111.2 % (CV=3.23 %, n=5) was obtained for 200 mg/mL.
Standard stability for 8 days was not proven (stored at 2 – 8 ºC). No results are reported.
Extract stability for 7 days was not proven (stored at 2 – 8 ºC). No results are reported.

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, 10 mg/mL and 200 mg/mL over three stability trials.
The overall procedural recovery results of the various validations and stability trials were assessed. Due to the greater variability found in the LC-MS/MS analysis, an acceptable range of 80 % to 120 % was used.

TRIAL 1 (2 mg/mL AND 200 mg/mL)
At 2 mg/mL, the 0 hour results were initially >10 % from the nominal concentration. Two other time points were >15 % from nominal therefore this concentration was repeated in Trial 2. However following re-calculation of results, the 0 and 4 hour results were subsequently shown to be acceptable. The Day 2 results were both >10 % from 0 hour therefore they did not meet the criteria for stability. The Day 15 results were not reported due to failed linearity and standard comparison. The CV was <5 % for all time points confirming the homogeneity of the formulation. Stability was confirmed for 4 hours ambient temperature storage and 22 days refrigerated storage.

At 200 mg/mL, stability was confirmed for 4 hours ambient temperature storage and 22 days refrigerated storage. Stability was not confirmed for 15 days storage as these results could not be reported due to a failed standard comparison. The CV was <5 % for all time points confirming the homogeneity of the formulation except for the 2 hour ambient temperature and the Day 22 refrigerated samples. The 200 mg/mL stability experiment was repeated in Trial 3.

TRIAL 2 (2 mg/mL and 10 mg/mL)
Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for 4 hours, and on re-suspension following refrigeration for up to 15 days. Stability was confirmed for 2 days at ambient temperature storage and 15 days refrigerated storage at 10 mg/mL.
Stability at 2 mg/mL was confirmed for 4 hours ambient temperature storage and 15 days refrigerated storage. The Day 2 samples at 2 mg/mL could not be reported as only one recovery sample was acceptable.

At each time-point, the mean analysed concentration for the samples remained within 7 % of the initial time zero value and the coefficient of variation was less than 4 %. Accurate preparation of the formulation was confirmed at 10 mg/mL. This concentration is sufficient for this study where the lowest dose was 20 mg/mL. At 2 mg/mL, the initial concentration was 15 % from nominal, therefore accurate preparation was not confirmed.

TRIAL 3 (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 15 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 3 %. The exception was the Day 8 refrigerated samples which had a mean +15.3 % of the initial time zero value and the coefficient of variation was 5.3 %. This result is superseded by the Day 15 result and is considered to be due to analytical error. For Trial 1, all the recoveries were within the acceptance limits (80 – 120 %).
For Trial 2, all recoveries were within acceptance limits (80 – 120 %) except for one 2 mg/mL recovery on Day 2.

For Trial 3, the recovery samples analysed on Day 0 and Day 2 were within the acceptance limits of 80 - 120 %. Recoveries on Days 8 and 15 were above the upper acceptance limits but were accepted as being representative of the recovery of the method on these occasions as the stability samples and recovery samples were similarly high relative to the nominal concentrations. Stability samples were acceptable after correcting for the high recovery results.

CONCENTRATION OF DOSE FORMULATIONS
The mean concentrations were within applied limits +10/-15 %, confirming the accuracy of formulation except for the Week 4 Group 3 samples. Precision, measured by % difference from the mean, was acceptable for all samples except Week 1 Groups 2 and 4 and Week 4 Group 3.

For Week 1, the recovery samples at 20 mg/mL and 60 mg/mL were initially above the upper acceptance limit and all recoveries including those at 200 mg/mL were re-diluted. The re-diluted results came out in the range 70.7 % to 83.5 %. When corrected for these recovery results, all the samples were acceptable. The low recovery results are explained by the failure of the associated standard comparison and therefore are not reported. The original results were accepted and are reported as the recovery results were representative of the extraction efficiency of the method on this occasion. The analytical evidence suggests that the Week 1 formulations were prepared correctly. For the Group 1 samples, peaks were observed at a similar retention time to the test item. Responses were below the response of the lowest linearity standard and are reported as not quantifiable (NQ). These responses were not investigated at the time.

For one of the Week 4 Group 3 samples one result was acceptable but the second was -35.5 % from nominal. In error this was not investigated, but the low result was thought to be due to an analytical error with the particular sample rather than an error in the formulation preparation as this was the only sample in this analysis which was outside the acceptance criteria. The recovery samples for Week 4 were all acceptable.

CONCLUSION
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, repeatability, method accuracy and precision. Standard and extracted solution stability were not established.

Homogeneity and stability were confirmed for the test material in corn oil formulations at nominal concentrations of 2 mg/mL, 10 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 4 hours, ambient temperature storage for 2 days (10 mg/mL and 200 mg/mL) and refrigerated storage for up to 15 days (10 mg/mL and 200 mg/mL) and 22 days (2 mg/mL).

The mean concentrations of the test material in test formulations analysed for the study were within +10/-15 % of nominal concentrations, confirming accurate formulation. The exception was the Group 3 samples for Week 4 where one result was acceptable and one result was low. The low result was thought to be due to an analytical error rather than an error in the formulation preparation. In the Week 1 Group 1 samples a response was observed in chromatograms at the retention of the test material, this was below the lowest linearity standard and is reported as NQ.
Duration of treatment / exposure:
A minimum period of four weeks followed by a two-week recovery period.
Cessation of treatment for the recovery phase animals started on the day of necropsy of animals allocated to the main study.
Frequency of treatment:
Once daily at approximately the same time each day.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 animals per sex per dose for the main study.
5 animals per sex for the high dose and control group in the recovery phase.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses used in this study, 100, 300 and 1 000 mg/kg/day, were selected in conjunction with the Sponsor.
An acute oral toxicity study showed that the acute median lethal oral dose (LD50) to rats of the test material was demonstrated to be greater than 2000 mg/kg. In a 14-day repeat-dose dose range finding study, doses of 100, 300 and 1000 mg/kg/day were well tolerated and did not result in any mortality or signs of systemic toxicity.
Based on this information, it was considered appropriate to investigate a high dose level of 1000 mg/kg/day (the limit dose for this study type). The intermediate and low dose levels were selected to assess any dose-responsiveness of any test substance-related finding.
- 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: 2 week recovery period (14 days).

Examinations

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’.
Clinical observations were presented for each animal that showed signs, providing detail of type of sign, week of occurrence and information on the duration of the sign applicable. There were no signs associated with dosing seen.

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
Daily during the treatment period, detailed observations were recorded at the following times in relation to dose administration:
- During Week 1 of treatment: Pre-dose, 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: Pre-dose, and one to two hours after completion of dosing of all groups.
- Recovery Period: No treatment-related signs were observed, so recovery phase observations were not required.

Detailed Physical Examination 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 for main study males and recovery phase animals and on Day -10 and -3 before treatment commenced for main study females, on the day that treatment commenced (Week 0), weekly throughout the study (last scheduled body weight recorded on Day 28 for the main study animals and Day 14 for the of recovery phase animals) and before necropsy.
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 for main study males and recovery phase animals and for Day -10 to Day -3 and Day -3 to Day -1 for main study females and for each week throughout the study (recorded on Day 29 during Week 4 of treatment and on Day 14 during Week 2 of recovery before overnight collection of urine).
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.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
Fluid intake was assessed by daily visual observation.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
Blood samples were collected on day 29 from all main study animals after an overnight withdrawal of food.
Blood sampling was performed on the morning after overnight collection of urine for females, and on the afternoon after overnight collection of urine for males. 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 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer: 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).

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 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.
The most common morphological changes, anisocytosis, micro/macrocytosis and hypo/hyperchromasia, were recorded according to the following convention:
- No abnormalities detected
+ Slight
++ Moderate
+++ Marked

CLINICAL CHEMISTRY: Yes
Blood samples were collected on day 29 from all main study animals after overnight withdrawal of food.
Blood sampling was performed on the morning after overnight collection of urine for females, and on the afternoon after overnight collection of urine for males. 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: Alkaline phosphatase (ALP), 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 Ratio = 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.
Group means and standard deviations are presented for volume, pH, specific gravity, protein, creatinine, glucose and electrolytes only.

NEUROBEHAVIOURAL EXAMINATION: Yes
- 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.


IMMUNOLOGY: No
Sacrifice and pathology:
METHOD OF KILL
Carbon dioxide asphyxiation with subsequent exsanguination. Main study animals were killed following four weeks of treatment. Recovery animals were killed following four weeks of treatment and two weeks of recovery.

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.
Main study animals were killed following four weeks of treatment. Recovery animals were killed following four weeks of treatment and two weeks of recovery.

ORGAN WEIGHTS
For bilateral organs, left and right organs were weighed together, unless specified. 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 killed at a scheduled interval.
- Abnormalities: only All main study animals of Groups 2 and 3 and all recovery phase animals killed at a scheduled interval.
- Testes and epididymis All main study males of Groups 2 and 3 and all Recovery phase males.
- Routine staining: Sections were stained with haematoxylin 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.

The following tissues, which were considered to exhibit a reaction to treatment at the high dose, were examined for all main study and recovery animals:
- Testes and epididymis
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

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs considered to be related with treatment and there were no signs associated with dosing.
Mortality:
no mortality observed
Description (incidence):
There were no deaths throughout the study period.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was no clear effect of treatment upon body weight.
The body weight gains of males receiving the test material at 100, 300 or 1 000 mg/kg/day during the 4-week treatment period (Week 0-4) were slightly higher than that of the Control, with statistical significance achieved for males at 1 000 mg/kg/day. However, due to the small group size, effect on one sex only, and absence of a dose-response, no effect of treatment is inferred.
Body weight gains of males and females which had received the test material at 1 000 mg/kg/day were similar to that of the Controls over the two-week recovery period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was considered 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):
Increased water consumption [visual assessment] was observed sporadically throughout the study, however, there was no consistency in the days observed or animals affected. It was therefore considered that there was no effect of treatment and consequently no quantitative measurements were performed.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The haematological examination performed in Week 4 did not indicate any toxicologically significant difference from controls. All inter-group differences from control, including those that attained statistical significance, were minor, confined to one sex or lacked dose-relationship and were therefore attributed to normal biological variation. Such differences included:
- For males receiving the test material at 1 000 mg/kg/day, mean cell haemoglobin concentration was slightly but statistically significantly higher than that of the Control, while for females receiving the test material at 100 mg/kg/day, mean cell haemoglobin concentration was slightly but statistically significantly lower than that of the Control.
- For males receiving the test material at 300 or 1 000 mg/kg/day, neutrophil counts were slightly lower than that of the Control (this change did not attain statistical significance) and activated partial prothrombin times were significantly shorter than that of the Control.
For females receiving at 1000 mg/kg/day, activated partial prothrombin times were significantly longer than that of the Control.
- For males receiving the test material at 100 mg/kg/day only, basophil counts were slightly but significantly higher than that of the Control.
Few additional variations from control values were recorded for females receiving the test material however, none of these slight differences attained statistical significance and in most cases no dose-relationship was apparent. Therefore, no effect of treatment is inferred.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The biochemical analysis of blood plasma in Week 4 did not indicate any toxicologically significant difference from controls.
All inter-group differences from control, including those that attained statistical significance, were minor, confined to one sex or lacked dose-relationship and were therefore attributed to normal biological variation. Such differences included:
- For males receiving the test material at 100, 300 or 1 000 mg/kg/day, triglyceride concentrations were slightly but not statistically significantly lower than that of the Control. There was a high degree of individual variation across the groups, including the Control group.
- For females receiving the test material at 100, 300 or 1 000 mg/kg/day, glucose concentrations were slightly and statistically significantly lower than that of the Control. A dose response was not apparent, and there was a high degree of individual variation across the groups, including the Control group.
- For females receiving the test material at 1 000 mg/kg/day, total protein and albumin concentrations were slightly but statistically significantly higher than that of the Control.
In the absence of findings similar in both sexes or any other effects of treatment with the test material, these changes are considered not to be of toxicological significance.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
The analysis of the urine in Week 4 did not indicate any toxicologically significant difference from controls.
For females receiving 1 000 mg/kg/day, the total potassium levels were significantly lower than that of the Control, however, in the absence of findings similar in both sexes (total potassium level for males receiving 1 000 mg/kg/day were slightly increased) or any other effects of treatment with the test material, this change was considered not to be of toxicological significance.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There was no effect of treatment on the organ weights of males or females receiving the test material at 100, 300 or 1 000 mg/kg/day at the end of the 4-week treatment period.
After the 2 week recovery period, organ weight data of males previously receiving the test material at 1 000 mg/kg/day showed slightly higher adjusted liver, and lower adjusted prostate and seminal vesicles and testes weights when compared to Controls. In the absence of any similar findings following 4 weeks of treatment, any relationship to previous treatment is considered unlikely.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- Animals Killed After 4 Weeks of Treatment
The macroscopic examination performed after 4 weeks of treatment revealed no test item related lesions.
The incidence and distribution of all findings were consistent with the common background seen at these laboratories.

- Animals Killed After 2 Weeks of Recovery
The macroscopic examination performed after 2 weeks of recovery revealed no test item related lesions. One male previously treated at 1 000 mg/kg/day was found to be monorchid (i.e., unilateral absence of the testis and epididymis) at necropsy. Although not a common background finding, this was considered to be a congenital change, unrelated to previous treatment with the test material.
All other findings were similar to the background of changes commonly seen at these laboratories.
Neuropathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
SENSORY REACTIVITY AND GRIP STRENGTH
Sensory reactivity and grip strength in Week 4 of treatment was considered unaffected by the administration of test material.

MOTOR ACTIVITY
Motor activity in Week 4 was considered unaffected by treatment.
Some intergroup differences were apparent, but were considered not to indicate an effect of treatment.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
- Animals Killed After 4 Weeks of Treatment and 2 Weeks of Recovery
Hypospermatogenesis and associated reduction/absence of sperm in the epididymides were seen in two males treated at 1 000 mg/kg/day and one male treated at 300 mg/kg/day. Although a dose-effect seemed to be present, in affected animals the seminiferous tubules had all cellular stages of the spermatogenic cycle apart from a partial depletion in elongating spermatids, which, as described in the literature (OECD Guidance Document No. 106), is not rare in animals younger than 10 weeks of age. Furthermore, a genuine test item related change would not have resolved over the 2 weeks recovery period and in this study no findings were seen in the testes and epididymides after 2 weeks off treatment. In conclusion, the findings in the testes and epididymides were considered to be related to sexual immaturity of males on study, and not an effect of treatment with the test material.
The incidence and distribution of all other findings were consistent with the common background seen at these laboratories.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Details on results:
Oral administration of the test material to Han Wistar (RccHan™;WIST) rats for 4 weeks at doses up to 1 000 mg/kg/day was well tolerated with no adverse effect of treatment.
There were no clinical signs nor any signs related to dosing, and there was no effect on sensory reactivity, grip strength or motor activity. Bodyweight gain was considered unaffected by treatment with the test material, and there was no effect of treatment with the tes material at 100, 300 or 1000 mg/kg/day on the food consumption of males or females. The haematological examination and biochemical analysis of blood plasma performed in Week 4 did not indicate any toxicologically significant difference from controls, and the analysis of the urine in Week 4 did not indicate any toxicologically significant difference from controls. There were no test item related organ weights changes and no findings seen at macroscopic or microscopic examination after 4 weeks of treatment or 2 weeks of recovery.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test material related effects

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study, the No-Observed-Adverse-Effect-Level (NOAEL) was considered to be 1000 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.

Three groups, each comprising five male and five female RccHan™WIST rats, received the test material at doses of 100, 300 or 1 000 mg/kg/day for 28 consecutive days. A similarly constituted control group received the vehicle, corn oil, at the same volume dose as the treated groups. A further five male and five female rats were assigned to each of the control and high dose groups. These animals were treated for four weeks, followed by a two-week 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, hematology (peripheral blood), blood chemistry, urinalysis, organ weight, macropathology and histopathology investigations were undertaken.

There were no signs associated with dosing or clinical signs at weekly physical examination and no findings at the sensory reactivity, grip strength or motor activity assessments. There was no toxicologically significant effect upon body weight, and food or water intake.

There were no toxicologically significant findings at the hematology, blood chemistry and urinalysis investigations performed in Week 4.

There was no treatment-related effect on organ weights and no treatment-related macroscopic or microscopic findings after 4 weeks of treatment or following 2 weeks of recovery.

It is concluded that oral administration of the test material to Han Wistar (RccHan™;WIST) for 4 weeks at dosages up to 1000 mg/kg/day was well tolerated with no adverse effect of treatment. Consequently, the No-Observed-Adverse-Effect-Level (NOAEL) was considered to be 1 000 mg/kg/day.