Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 December 2014 to 7 July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 407 without any deviation.
Justification for type of information:
Information as to the availability of the in vivo study is provided in 'attached justification'.

Data source

Referenceopen allclose all

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3050 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
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
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on 01 July 2014/ signed on 07 October 2015)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Colorless liquid
- Storage condition of test material: Stored at 2-8 °C temperature and protected by Nitrogen.
Specific details on test material used for the study:
- Purity test date: 04 November 2014
- Storage condition of test material: Refrigerated at 4°C in the dark under nitrogen

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies.
The Crl:CD(SD) strain was used because of availability of historical control data at this laboratory.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 35-41 days
- Weight at study initiation: Males: 160-199 g; Females: 128-163 g
- Housing: Five animals/sex/cage (main study and recovery) in polycarbonate cages with a stainless steel mesh lid
- Diet: Harlan Teklad 2014 rodent diet, ad libitum; Non-restricted (removed overnight before blood sampling for haematology or blood chemistry and during the period of urine collection).
- Water: Potable water from the public supply, ad libitum
- Acclimation period: 6 days

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

IN-LIFE DATES: 15 December 2014 to 7 July 2015

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral gavage route of administration was chosen to simulate a potential route of accidental exposure and is the recommended route in the test guideline.
Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Method of preparation: Starting with the lowest concentration, approximately 50% of the final volume of vehicle was added to the required amount of test material and magnetically stirred until all of the test material was thoroughly mixed. The remaining amount of vehicle was added to make up to the required total volume. The formulation was mixed using a magnetic stirrer to produce a homogenous suspension. The remaining concentrations were formulated in ascending order using the same method.
Frequency of preparation: Weekly
Storage of preparation: Refrigerated (nominally 4 °C)

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw/day

DOSE VOLUME: 5 mL/kg bw/day
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 1 and 200 mg/mL were analysed to assess the stability and homogeneity of the test substance in the liquid matrix.
Achieved concentration: Samples of each formulation prepared for administration in Weeks 1 and 4 of treatment were analysed for achieved concentration of the test substance.

Results:
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limits of detection and quantification, linearity of detector response, repeatability, method accuracy and precision. The homogeneity and stability was confirmed for test item in corn oil formulations at nominal concentrations of 1 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage for 1 day and refrigerated storage for up to 15 days. The mean concentrations of test item in test formulations analysed for the study were within 6% of nominal concentrations, confirming accurate formulation. The precision of individual results from mean values was less than 5%, confirming precise analysis.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily at approximately the same time each day.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Main study: 5/sex/dose (0, 100, 300 and 1000 mg/kg bw/day)
Recovery phase: 5/sex/dose (0 and 1000 mg/kg bw/day)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected after consultation with the Sponsor and were based on data from a 7-day preliminary toxicity study (Huntingdon Life Sciences Study Number HIK0057). In the preliminary study, test item ST 05 C 14 was administered by gavage to 3 male and 3 female CD rats at doses of 250, 500 or 1000 mg/kg bw/day for 7 days. Animals were tolerated with no significant clinical signs, no effects on body weight or food consumption or any treatment-related deaths. Salivation and/or chin rubbing, which was observed shortly after dosing, predominantly for animals receiving 500 or 1000 mg/kg/day is commonly encountered in studies where the test substance is administered orally. The chin rubbing was considered to be secondary to the excessive salivation. Adjusted liver weights were marginally high in animals given 500 or 1000 mg/kg/day. This is a common response following administration of a xenobiotic at a high concentration and in the absence of any macropathological change observed in the liver this finding is considered not adverse in the context of this study. None of the findings observed would preclude administration of dosages up to 1000 mg/kg bw/day in a 28-day repeat dose study (Huntingdon Life Sciences Study No. HIK0061); therefore 1000 mg/kg bw/day (the limit dose) is considered to be a suitable high dose. Doses of 100 and 300 mg/kg bw/day were selected for the low and intermediate levels.
- Rationale for animal assignment: Randomly allocated on arrival.
- Post-exposure recovery period in satellite groups: 14 days
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants. During the acclimatisation and recovery periods, observations of the animals and their cages were recorded at least once per day.

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

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded four days before treatment commenced, on the day that treatment commenced (Day 1), weekly throughout the study and on the day of necropsy prior to despatch from the animal facility.

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 (over a four day period) and for each week throughout the study.

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

OPHTHALMOSCOPIC EXAMINATION: No

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

URINALYSIS: Yes
- Time schedule for collection of urine: Week 4 (all main study animals); Recovery Week 2 (all recovery phase animals)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes; Animals were placed in an individual metabolism cage, without food or water.
- Parameters checked:
Using manual methods: Clarity and Colour - by visual assessment, Volume - using a measuring cylinder,
pH - using a pH meter, Specific gravity - by direct refractometry using a SG meter
Using Multistix reagent strips, interpreted using a Clinitek®500 instrument: Ketones, Bilirubin/bile
pigments, Urobilinogen, Blood pigments
Using a Roche P Modular analyser: Protein, Creatinine, Glucose, Sodium, Potassium, Chloride
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 below:
Epithelial cells, Leucocytes (WBC), Erythrocytes (RBC), Casts and Other abnormal components
The slide was also examined for abnormalities in spermatozoa and crystals.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Detailed physical examination and arena observations were performed on each animal before the treatment period commenced and during each week of treatment and recovery. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities.
- Battery of functions tested: sensory activity / grip strength / motor activity
Sensory reactivity and grip strength: Sensory reactivity and grip strength assessments were performed (before dosing) on all main study animals in Groups 2 and 3 and all recovery animals in Groups 1 and 4 during Week 4 of treatment.
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 animals in Groups 1 and 4 was measured using a Rodent Activity Monitoring System (Version 2.0.5), with hardware supplied by Pearson Technical Services and software developed and maintained by Envigo.

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

GROSS PATHOLOGY: Yes; after a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

ORGAN WEIGHTS: For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for main study and recovery animals killed at scheduled intervals.

HISTOPATHOLOGY: Yes
Fixation: Tissues were routinely preserved in 10 % Neutral Buffered Formalin with the exception of those detailed below:
Testes: In modified Davidson’s fluid; Eyes: In Davidson’s fluid.
Histology:
Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Full List: Main study animals of Groups 1 and 4 killed at a scheduled interval.
Abnormalities, liver (males and females), kidneys and thyroids (males only): All main study animals of Groups 2 and 3 and all recovery phase animals killed at a scheduled interval.
Routine staining: Sections were stained with haematoxylin and eosin
Light microscopy: Tissues preserved for examination were examined as follows:
Scheduled kill:
Main study: All animals of Groups 1 and 4 (all specified in Table 7.5.1/1)
All animals of Groups 2 and 3 (Abnormalities, liver (males and females), kidneys and thyroids (males only))
Recovery: All animals of Groups 1 and 4 (Abnormalities, liver (males and females), kidneys and thyroids (males only))
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:
Liver - males and females; Kidneys - males only; Thyroids - males only
Other examinations:
None
Statistics:
See "Any other information on materials and methods incl. tables"

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
- There were no adverse clinical signs observed at the routine weekly examination that were considered related to treatment.
- Signs associated with the dosing procedure were observed in the first week of treatment and comprised salivation observed soon after dosing in animals that received 1000 mg/kg bw/day and, to a lesser extent, in animals that received 300 mg/kg bw/day. Chin rubbing was observed on Day 6 in one female that received 1000 mg/kg bw/day.
CONCLUSION: No adverse effects
Mortality:
no mortality observed
Description (incidence):
No mortality was observed.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- In the first week of treatment (Days 1-8) females that were dosed with 1000 mg/kg bw/day gained more body weight than the controls (x1.3 control; attaining statistical significance), however, increased body weight gain was not evident for the remainder of the treatment period for this group. Body weight gains for males were similar to control during the treatment period.
- Body weight gain during the first week of the recovery period (Days R1-R8) was high for males previously treated with 1000 mg/kg bw/day, when compared with the controls (x1.8 control; attaining statistical significance). However, in the second week of the recovery period the body weight gains of these males were similar to controls. In females previously treated with 1000 mg/kg bw/day overall body weight gain in the recovery period was slightly low when compared with the controls (x0.64 control); this change was not statistically significant.
CONCLUSION: No adverse effects
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption was not affected by treatment.
The food consumption was variable in all the treatment weeks. In the males there was a trend for all groups of test item treated animals to eat more food than the control group but no dose response was evident. This trend continued in the previously treated males during the recovery phase. It was considered that the variability in food consumption was background variation and was not related to treatment.
CONCLUSION: No adverse effects
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
In Week 3, an increase in water consumption was observed amongst males and females receiving 1000 mg/kg bw/day when compared with Controls.
CONCLUSION: No adverse effects
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
- Overall, there were no adverse, treatment-related changes in the haematology parameters.

- Haematological examination after 28 days of treatment revealed statistically significantly higher platelet numbers in males treated at 100, 300 or 1000 mg/kg bw/day compared with control animals. However, this finding was considered to be due to a slightly low control value of 845 x10^9/L [historical control data range: 869 - 1466 x10^9/L], and as a similar effect of treatment was not evident in females the finding in males was considered unlikely to be treatment-related. After the two week recovery period the values for platelet numbers in males that had received 1000 mg/kg bw/day were similar to the control group; with control values improved and with the background control data range.

- When compared with the control animals, statistically significantly lower mean cell haemoglobin concentration was noted in males treated at 300 or 1000 mg/kg bw/day. There was no dose relationship and in the absence of an effect of treatment of any of the other red cell indices this difference from control was not considered to be treatment-related. At the end of the recovery period the values for mean cell haemoglobin in males that had previously received 1000 mg/kg bw/day were comparable to the control group.

- Two females that received 1000 mg/kg bw/day had shorter activated partial thromboplastin times compared with the controls (13.9 and 11.4 seconds, respectively, compared with 15.8 seconds as the lowest clotting time in the control female group); comparison of the group mean values for activated partial thromboplastin times for the treated and control groups did not reveal statistically significant differences. There was no evidence of an effect on activated partial thromboplastin times in females previously treated with test item at the end of the recovery period.

- After the two week recovery period, males previously treated at 1000 mg/kg bw/day had decreased haemoglobin concentration and erythrocyte counts, and increased red cell distribution width, when compared with the control group with all of the changes achieving statistical significance. These parameters were not affected after 28 days of treatment and consequently these variations from control were considered unrelated to the previous administration of the test material.

CONCLUSION: No adverse effects
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- Examination of the plasma after 28 days of treatment revealed, when compared with the controls, statistically significantly lower mean plasma urea concentration in females receiving 1000 mg/kg bw/day. At the end of the recovery period, the plasma urea concentrations in females that had previously received 1000 mg/kg bw/day were comparable to control.

- Higher than control albumin and consequently higher total protein concentrations were apparent in males that received 1000 mg/kg bw/day, which resulted in a lower mean albumin:globulin ratio in this group of animals; these changes all achieved statistical significance. After 2 weeks without treatment there was evidence of recovery in this high dose male group with albumin and total protein values comparable to the control group however, the mean albumin:globulin ratio remained statistically significantly lower than controls in this group. A statistically significantly lower mean albumin:globulin ratio was noted at the end of the recovery period for females treated with test item, but there was no such finding at the end of the treatment period.

- Statistically significant group mean increases in plasma cholesterol and triglyceride concentrations were reported for females that received 1000 mg/kg bw/day. No difference from control was apparent for triglyceride concentrations in the previously test item treated females at the end of the recovery period. Cholesterol concentrations improved following 2 weeks of recovery however mean concentrations in these females remained marginally high and achieved statistical significance. Plasma cholesterol concentrations were also statistically significantly high in 4 out of 5 males previously given 1000 mg/kg bw/day, an effect that was not observed at the end of the treatment period.

- Minor, but statistically significant, electrolyte changes (decreased chloride and increased calcium concentrations) were evident in males that received 1000 mg/kg bw/day. The effect on calcium concentration was still present after two weeks of recovery however, the magnitude of difference from the control group was reduced suggesting partial recovery of this electrolyte change. In addition, plasma chloride concentrations were slightly decreased in females that received 1000 mg/kg bw/day (statistical significance was not attained) and plasma phosphorus concentrations were slightly increased and achieved statistical significance; these differences were still apparent in the females previously given 1000 mg/kg bw/day at the end of the two week recovery period.

- After the two week recovery period, lower than control alkaline phosphatase activities were evident in males and females previously treated with test item (statistical significance only attained in the males). In the absence of a similar difference at the end of the treatment period, this change was considered to be attributable to natural variation, and unrelated to previous treatment.

CONCLUSION: No adverse effects
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
- Analysis of the urine after 28 days of administration revealed statistically significantly lower urinary pH amongst females treated at 100 or 300 mg/kg bw/day and in males and females treated at 1000 mg/kg bw/day when compared with control. Specific gravity was statistically significantly higher than the control group, in all female test item treated groups.

- When compared with the controls, statistically significantly higher urinary total sodium and total chloride were reported in males that received 1000 mg/kg bw/day. Total sodium was statistically significantly lower than control for all groups of test item treated females; this finding was without dose relationship (the lowest result being recorded in the low dose group) and was considered not an effect of treatment in the females.

- Higher than control urinary glucose was recorded for all test item treated male groups, achieving statistical significance at 1000 mg/kg bw/day; however, the control mean was skewed by low values in two animals. The highest values for urinary glucose were observed in males that received 300 or 1000 mg/kg bw/day, with the magnitude of change between these groups suggesting a dose-related response. It was considered that this small increase in total glucose in these few animals was likely to be related to treatment but was not considered to be adverse.

- Microscopic evaluation revealed the presence of unidentified crystals/clumps of material in the sediment of two males given 300 mg/kg bw/day and in two male animals given 1000 mg/kg bw/day. No evidence of unidentified crystals/clumps of material was identified at the end of the recovery period in males, or in any female animals.

- After 2 weeks of recovery, urinary pH (males and females) and total sodium and total chloride concentrations (in males) were comparable with the control group, demonstrating full recovery. In previously treated test item females specific gravity remained high in two of the animals and, in previously treated males, glucose remained slightly high when compared with the control animals, although statistical significance was not attained for either of these changes. When compared with the controls, specific gravity was statistically significantly low in previously treated test item males but in the absence of a similar difference at the end of the treatment period, this change was considered to be attributable to natural variation, and unrelated to previous treatment.

CONCLUSION: No adverse effects
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
- Sensory reactivity and grip strength assessments did not reveal any adverse responses to treatment with test item.
- Motor activity assessments did not reveal any adverse responses to treatment with test item.
CONCLUSION: No adverse effects
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- Body weight adjusted liver weight was higher than control in males and females given 1000 mg/kg bw/day (x1.2 and x1.3 control for males and females, respectively). There was evidence of recovery in the females, but in the males the liver weights remained high (x1.2 control) achieving statistical significance.
- Higher than control body weight adjusted kidney weights were evident in males given 300 mg/kg bw/day and in males and females given 1000 mg/kg bw/day (with statistical significance achieved only in the males). There was full recovery in the kidney weights in both males and females, with the absolute and adjusted weights similar to the controls; no statistical significance was attained.
CONCLUSION: No adverse effects
Gross pathological findings:
no effects observed
Description (incidence and severity):
- There were no macroscopic abnormalities detected at scheduled termination after four weeks of treatment or after two weeks of recovery that were attributable to treatment with test item.
- The incidence and distribution of all findings were consistent with the common background findings observed at these laboratories in this species and strain.
CONCLUSION: No adverse effects
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Changes related to treatment with test item were observed in the liver, kidneys and thyroids, mainly in the males.
- Liver: Centrilobular hypertrophy was observed in one female treated with 300 mg/kg bw/day, and in five males and three females treated with 1000 mg/kg bw/day. Following 2 weeks without treatment; treatment-related findings were still present in the liver of one male previously treated with 1000 mg/kg bw/day. This is indicative of complete recovery in the females and partial recovery in males.
- Kidney: Minimal hyaline droplets were recorded in three males treated with 300 mg/kg bw/day and in three males treated with 1000 mg/kg bw/day.
- Thyroid: Minimal follicular cell hypertrophy was recorded in all male groups including controls, with 4/5 animals affected at 300 and 1000 mg/kg bw/day. At 100 mg/kg bw/day the change was considered likely to be background and unrelated to treatment, as the incidence was only slightly above control levels and some level of variation is normal in thyroids of young males.
Changes previously recorded in the thyroids and kidneys of males receiving 300 and 1000 mg/kg bw/day were no longer apparent after 2 weeks of recovery.
- All other histological changes were considered to be unrelated to treatment.
CONCLUSION: No adverse effects
Other effects:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed
Remarks on result:
other: none of the findings were considered adverse in this study.

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Discussion:

The systemic toxic potential of test item was assessed by oral administration to Crl:CD (SD) rats over a period of four weeks followed by a two week recovery period. Doses up to 1000 mg/kg bw/day were tolerated with treatment-related changes observed mainly in clinical pathology, kidney and liver weights and microscopic changes within the liver (centrilobular hypertrophy), kidneys (hyaline droplets) and thyroid (follicular cell hypertrophy).

Histopathological examination identified the liver as a target organ. There was evidence of adaptive changes in the liver at 1000 mg/kg bw/day (males and females) and to a lesser extent at 300 mg/kg bw/day (females). These changes included increased cholesterol levels, increased liver weights and centrilobular hypertrophy. Orally administered substances are metabolized by the liver, therefore increased activity levels in the liver are considered to be a normal adaptive response. There was evidence of full recovery in the females and partial recovery in the males and as these changes reflect an adaptive change they are not considered to be adverse.

The follicular cell hypertrophy in the thyroid observed in males at 300 or 1000 mg/kg bw/day might be linked to enzymatic induction in the liver, of which is a well-recognized rodent specific phenomenon. Hepatic microsomal enzyme induction in the liver can lead to an increased breakdown of thyroid hormones and consequent stimulation of the normal feedback control of the thyroid (Richardson and Klaasen, 2010). This finding is therefore considered secondary to the effect in the liver and, given that it is not relevant to humans, it is considered not to be an adverse finding.

In this study, follicular cell hypertrophy and centrilobular hypertrophy were observed in thyroid and liver respectively of the males treated with 1000 mg/kg bw/day, but only follicular cell hypertrophy of the thyroids was observed in males treated with 300 mg/kg bw/day. In males treated with 300 mg/kg bw/day, the thyroid effects are likely secondary to a low grade hepatocellular hypertrophy which was undetectable by light microscopy due to its minimal severity and the small number of animals in each group. Similarly, centrilobular hypertrophy of the liver was observed in females treated with 300 and 1000 mg/kg bw/day, but no changes were recorded in the thyroids. Although the correlation between thyroid and liver changes was not clear-cut at 300 mg/kg bw/day, the presence of both changes together in males treated with 1000 mg/kg bw/day is indicative of a causal relationship between the liver and thyroid changes.

In the kidney, males treated with 300 mg/kg bw/day or 1000 mg/kg bw/day showed accumulation of hyaline droplets in the proximal tubules. The accumulation of hyaline droplets is indicative of alpha2μ-globulin nephropathy, which is peculiar to mature male rats (De Rijk, 2003). This finding correlated with a slight increase of the kidney weights in this group of animals. Hyaline droplet production is not relevant to humans and, therefore, this finding is not a sign of adverse toxicity.

Minor disturbances in urinary pH, specific gravity (females only) and electrolytes were noted at the Week 4 investigation and in Week 3 a small increase in water consumption was observed amongst animals receiving 1000 mg/kg bw/day when compared with controls. At the end of the recovery period the urinary parameters in the previously treated animals were generally similar to control except for specific gravity which remained very high in two females previously given the test material. The aetiology of these findings is unknown and in the absence of any pathological findings in the kidney of both sexes these changes in urinary parameters are not considered to be adverse.

Salivation and chin rubbing, which were observed shortly after dosing during Week 1, are frequently observed signs in studies where the test material is administered by gavage and are considered to be typically related to the taste of the test-substance.

Other findings at 1000 mg/kg bw/day consisted of high bodyweight gain during the first week of treatment for females, reduced plasma urea (females), increased albumin and consequent total protein (males), plasma electrolyte disturbances (reduced chloride and increased calcium) in males, high urinary sodium and chloride (males). These findings were not consistent between the sexes, did not correspond with changes in any other parameter and generally showed reversibility, therefore, they were considered not to represent adverse toxicity.

As the findings on this study were considered to be adaptive, non-adverse and with evidence of reversibility the dose of 1000 mg/kg bw/day was considered to be the no-observed-adverse-effect-level (NOAEL) in this study.

Applicant's summary and conclusion

Conclusions:
Oral administration of test item to CD rats for 28 days at doses up to 1000 mg/kg bw/day were tolerated with treatment-related changes seen mainly in the plasma biochemistry and urinalysis parameters, some of which were considered indicative of adaptations of metabolism/excretion in the liver and kidneys, and were accompanied by small increases in liver and kidney weight. Microscopic changes were evident in the liver (centrilobular hypertrophy), kidneys (hyaline droplets) and thyroid (follicular cell hypertrophy). None of the findings were considered adverse and, therefore, the NOAEL was concluded to be 1000 mg/kg bw/day.
Executive summary:

In a repeated dose toxicity study performed in accordance with OECD test guideline No. 407 and in compliance with GLP, test item was administered by gavage to 5 male and 5 female Crl:CD(SD) rats at doses of 100, 300 or 1000 mg/kg bw/day. A similarly constituted control group received the vehicle, corn oil, at the same volume-dose. A further five male and five female rats were assigned to each of the control and high dose groups. These animals were treated for 28 days, followed by a 14 day period without treatment to assess the potential for any treatment-related change to recover. During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, water consumption (visually assessed), haematology (peripheral blood), blood chemistry, urinalysis, organ weight, macropathology and histopathology investigations were undertaken.

 

There were no deaths on study and no clinical signs were observed at the routine weekly examinations that were considered related to treatment.

 

Sensory reactivity, grip strength and motor activity assessments did not reveal any response to treatment with test item.

 

Body weight gain for females that received 1000 mg/kg bw/day was high (x1.3 Control; achieving statistical significance) during Week 1 but thereafter was comparable to the control group. Body weight gain of male rats was not affected during treatment but in the first week of the recovery period the body weight gain for males previously treated with 1000 mg/kg bw/day was high (x1.8 Control; achieving statistical significance) when compared with the controls. In females previously treated with 1000 mg/kg bw/day overall body weight gain in the recovery period was slightly low (x0.64 Control; statistical significance was not achieved) when compared with the controls.

 

Food consumption was not affected by treatment.

 

Haematological examination did not reveal any changes that were clearly attributable to administration with test item.

 

After 28 days of treatment, biochemical analysis of the plasma revealed low urea concentration in females that received 1000 mg/kg bw/day (resolved at the end of the recovery period); higher than control albumin and consequently higher total protein concentrations in males that received 1000 mg/kg bw/day (resulting in a lower mean albumin:globulin ratio in these animals: the A:G ratio remained low at the end of the recovery period); decreased chloride and increased calcium concentrations in males that received 1000 mg/kg bw/day with the effect on calcium still present after two weeks of recovery; decreased plasma chloride concentrations and increased plasma phosphorus concentrations in females that received 1000 mg/kg bw/day (these differences were still apparent in at the end of the two week recovery period). At the end of the recovery period a low mean albumin:globulin ratio was noted for females previously treated with test item (no effect noted in high dose females at the end of the treatment period) and, similarly, cholesterol concentrations were high in males and females that received 1000 mg/kg bw/day, an effect also not observed at the end of the treatment period (this was previously observed only one female that received 1000 mg/kg bw/day).

 

Urinalysis investigations after 28 days of treatment revealed the following changes that were considered to be related to treatment: low pH in all treated female groups and in males that received 1000 mg/kg bw/day, marginally high specific gravity in all female treated groups and high total sodium and total chloride levels in males that received 1000 mg/kg bw/day. Higher than control urinary glucose was recorded for all treated male groups; however, the control mean was skewed by low values in two animals. Microscopic evaluation revealed the presence of unidentified crystals/clumps of material in the sediment of two males given 300 mg/kg bw/day and in two animals given 1000 mg/kg bw/day; however, this was not observed at the end of the recovery period. After 2 weeks of recovery, urinary pH (males and females) and total sodium and total chloride concentrations (in males) were comparable with control, demonstrating full recovery. Specific gravity remained slightly high in previously treated females and glucose levels remained slightly high in previously treated males when compared with the controls.

 

Body weight adjusted liver weight was higher than control in males and females given 1000 mg/kg bw/day. There was evidence of recovery in the females. Higher than control body weight adjusted kidney weights were evident in males given 300 mg/kg bw/day and in males and females given 1000 mg/kg bw/day; recovery was evident in both sexes.

 

There were no macroscopic abnormalities detected at scheduled termination after four weeks of treatment or after two weeks of recovery that were attributable to treatment with test item.

 

Histopathological examination following 28 days of treatment revealed minimal, non-adverse changes in the liver of both sexes, and kidneys and thyroids of the males. Centrilobular hypertrophy was observed in the livers of one female treated with 300 mg/kg bw/day and in five males and three females treated with 1000 mg/kg bw/day. Hyaline droplets were recorded in the kidneys of three males treated with 300 mg/kg bw/day and three males treated with 1000 mg/kg bw/day. Follicular cell hypertrophy of the thyroids was recorded in all male groups including controls, with a high incidence at 300 and 1000 mg/kg bw/day. At 100 mg/kg bw/day the change was considered likely to be background and unrelated to treatment, as the incidence was only slightly above control levels and some level of variation is normal in thyroids of young males. At the end of the two week recovery period centrilobular hypertrophy was still present in the liver of one male previously treated with 1000 mg/kg bw/day indicating partial recovery in this sex and complete recovery in the females. The thyroid and kidney changes had completely resolved.

These findings were not consistent between the sexes, did not correspond with changes in any other parameter and generally showed reversibility, therefore, they were considered not to represent adverse toxicity. As the findings on this study were considered to be adaptive, non-adverse and with evidence of reversibility the NOAEL was considered to be 1000 mg/kg bw/day.

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