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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Remarks:
The 13 weeks repeated dose toxicity study was conducted solely to comply with non-EU national registration requirement, and has been provided here in accordance with REACH, Article 22(1)e.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 13 February 2013 to 09 May 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The Han Wistar rat was selected as the test mode too satisfy regulatory requirements for repeat dose testing, because of the availability of historical background data in this species and proven suitability in toxicity studies.
Acceptable models which do not use live animals currently do not exist.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 5 - 6 weeks
- Weight at study initiation: 118-149 g for males and 103-142 g for females
- Fasting period before study: No
- Housing: Animals were housed 2 or 3 per cage by sex in polycarbonate cages (dimensions 61 x 43.5 x 24 cm) with stainless steel grid tops
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 days

DETAILS OF FOOD AND WATER QUALITY:
SDS Rat and Mouse (modified) No.1 Diet SQC Expanded (Special Diet Services, Essex,
UK) and water taken from the public water supply (Scottish Water, Edinburgh, Midlothian,
UK).


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23°C
- Humidity (%): 56 - 65%
- Air changes (per hr): 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The dose volume for each animal was based on the most recent body weight measurement.
The dose volume of was 10 mL/kg.
The doses were given using a syringe with attached gavage cannula.
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1% carboxymethylcellulose (aq)
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
Test substance dosing formulations were prepared based on a method established at the Test Facility at appropriate concentrations to meet dosage level requirements. The dosing
formulations were prepared weekly, stored at -20°C in the dark, and dispensed daily.


- VEHICLE
Identification: Carboxymethylcellulose sodium (Medium Viscosity)
Batch (lot) numbers: SLBB55612V
Description: White powder
Expiry dates: 09 Jan 2014
Supplier: Sigma – Aldrich, UK
Storage conditions: Ambient laboratory conditions (< 30ºC)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected from all dose groups on week 1, 6 and 12 for concentration and homogeneity analysis.
Duplicate samples were taken from the top, middle and bottom of each formulation (including control) prepared for dosing on Day 1 and during Weeks 6 and 12 (2 x 1 mL).
The samples were kept at ambient temperature up to 24 h prior to analysis and at -20°C for back up samples.
Samples were analysed within 1 week of preparation.
Duration of treatment / exposure:
at least 91 days
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 male and 10 female rats
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were based on a preliminary study carried out by Charles River (Charles River Study No. 521852). Dose level selection took into account the maximum tolerated dose in the test model.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: each day 2x, Posture/condition on first approach (animal undisturbed), checking for:
Prostration, Lethargy, Writhing, Circling, Breathing abnormalities, Gait abnormalities, Tremor, Fasciculation, Convulsions, Biting (of cage components or self mutilating), Vocalisations, Piloerection.
Latency (time to first locomotory movement), Level of mobility, Rearing, Grooming, Urination/defaecation, Arousal (level of alertness), Posture, tremor/convulsions, vocalisation, piloerection, Palpebral closure, Gait abnormalities


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: each day 2x

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded once during pretrial and daily during the dosing period. Body weights recorded during the dosing period have been reported weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
The quantity of food consumed by each cage of animals was measured and recorded once
during pretrial and weekly during the dosing period.

FOOD EFFICIENCY:
body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / Not specified
Water consumption was qualitatively monitored by visual inspection of the water bottles on a
weekly basis during the study.

OPHTHALMOSCOPIC EXAMINATION: Yes / No / Not specified
The eyes of all animals (including extras) were examined once during the pretrial period using an indirect ophthalmoscope after the application of mydriatic agent (1% Tropicamide, Mydriacyl®). Anterior, lenticular and fundus areas were evaluated. All control and high dose animals were also examined during Week 13.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 13
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: No
- How many animals: All
- Parameters checked:
Red Blood Cell Count Platelets, Haemoglobin Blood smeara, Haematocrit White Blood Cell Count ,Mean Cell Volume Neutrophils, Mean Cell Haemoglobin Concentration Lymphocytes, Mean Cell Haemoglobin Monocytes, Reticulocytes Eosinophils, Reticulocyte Count (absolute) Basophils, Red Cell Distribution Width Large Unclassified Cells.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13
- Animals fasted:No
- How many animals: All
- Parameters checked:
Urea Total Protein, Glucose Albumin, Aspartate Aminotransferase Globulin, Alanine Aminotransferase, Albumin/Globulin ratio, Alkaline Phosphatase Cholesterol, Creatine Phosphokinase Creatinine, Lactate Dehydrogenase Total Bilirubin, Sodium Calcium, Potassium Inorganic Phosphate Chloride.

URINALYSIS: Yes
- Time schedule for collection of urine: Week 13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked:
Microscopic evaluation of the spun deposit Glucose, Colour Bilirubin, Turbidity Ketones, Specific gravity Leukocytes, Volume Blood Pigments, pH Urobilinogen, protein


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once during pretrial and once during Week 12
- Dose groups that were examined: All
- Battery of functions tested: sensory activity / grip strength / motor activity / Landing foot splay

IMMUNOLOGY: No


Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Statistics:
• All analyses were two-tailed for significance levels of 5% and 1%. Males and females were analysed separately.
• All means are presented with standard deviations.
• Body weights, cumulative body weight gain, selected functional observation battery and motor activity, absolute organ weights, haematology, coagulation, clinical chemistry parameters and urinalysis were analysed initially by a one-way analysis of variance (ANOVA).
• Organ weights were also analysed by analysis of covariance (ANCOVA) on final body weight (Shirley, 1977). This statistical analysis provided Adjusted Org n Weight values.
• Summary values of organ to body weight ratios and food consumptions are presented but these were not analysed statistically.
• For all parameters evaluated initially by ANOVA or ANCOVA, Dunnett’s test was used to compare the control (Group 1) and treated groups, based on the error mean square in the ANOVA or ANCOVA. The Dunnett’s test was performed for all continuous data parameters, regardless of whether the initial ANOVA or ANCOVA was statistically significant.
• Micropathology incidence data were analysed using Fisher’s Exact Test. Findings with multiple severities were analysed using a Mann-Whitney U-test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
All signs encountered were considered to be typical for rats of the age and strain used and considered to be unremarkable. There was no evidence of a response to treatment.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
During Week 13, fibrinogen values for both males and females receiving 1000 mg/kg/day were statistically significantly higher than control. With no findings in remaining clotting parameters, or findings in reticulocytes and/or platelets, no toxicological importance is attached to the finding of increased fibrinogen.
Derived red cell parameters (mean corpuscular haemoglobin, mean corpuscular volume) were lower than control while red cell width higher for males receiving 250 mg/kg/day and 1000 mg/kg/day. The differences from control attained statistical significance for all but mean corpuscular haemoglobin for males receiving 250 mg/kg/day. In the absence of findings in primary red cell parameters, these differences were considered to be incidental to treatment.
Statistical differences in white blood cell, lymphocyte and basophil counts for males receiving 250 mg/kg/day, basophil count for female receiving 250 mg/kg/day and activated partial thromboplastin time for males receiving 50 mg/kg/day were, in each case considered not to be related to the administration as similar differences from control were not seen in higher dose group animals.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
During Week 13, alanine. and aspartate aminotransferase activities were statistically significantly higher than control for males receiving 1000 mg/kg/day. In addition, total protein for both males and females receiving 250 mg/kg/day and 1000 mg/kg/day and albumin and globulin for males and females receiving 1000 mg/kg/day were statistically significantly higher than control. Phosphate values were statistically significantly lower than control for females at all dose levels.
Higher plasma sodium levels for treated females dosed with the test item (all dose levels) were considered to be within normal control range (Cl 39-153 mmol/L). Lower aspartate aminotransferase activities in females dosed with the test item (all dose levels) were considered to reflect a high control value, as AST activities were increased in high dose males and the values for treated females were with one exception (Animal 75F with AST activity of 41 U/L) within the historical control range (AST 42-188 U/L). Statistical differences in lactate dehydrogenase in females receiving 25 mg/kg/day and chloride and calcium for males receiving 25 mg/kg/day were considered to be spurious.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
During Week 12, males receiving 1000 mg/kg/day had increased pools of urine compared with controls when in the observation arena. There was no evidence of an effect on urine parameters and this finding is considered not to be toxicologically significant. There was no effect of treatment apparent on any other FOB parameter.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Following 13 weeks of treatment, liver and kidney weights for males receiving 250 mg/kg/day and males and females receiving 1000 mg/kg/day were higher than control.
The differences attained statistical significance for absolute organ weight and when body weight was used as covariate in the analysis. When body weight was used in the analysis, liver weight for females receiving 250 mg/kg/day and kidney weight for females receiving 50 mg/kg/day and 250 mg/kg/day were statistically significantly higher than control, although for kidney weights the differences were not dose related.
Statistically significant differences in thyroid weight for females receiving 50 mg/kg/day and lung weight for females receiving 250 mg/kg/day and spleen weight (body weight used as covariate in the analysis) in males receiving 1000 mg/kg/day were considered to be spurious.
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Description (incidence and severity):
In the liver, chronic, minimal to moderate arteritis/periarteritis with a focal to multifocal distribution was noted in males given 250 or 1000 mg/kg/day. This finding was characterised by periarterial infiltration by mononuclear inflammatory cells and there was expansion of the adventitial part of the artery by fibrous tissue occasionally associated with cell debris. In the media, some vessels had only plump nuclei without any degenerative or necrotic changes, but in some others, disruption of elastic fibres, cell debris, necrosis and/or haemorrhage were noted. The endothelium was occasionally reactive. Arteritis/periarteritis was not observed in any females and was present only in the liver of treated males. This finding was considered to be related to test item administration, but the direct or indirect nature of the effect cannot be established.
Minimal to mild centrilobular hepatocellular hypertrophy was observed in two females given 250 mg/kg/day, and one male and all females given 1000 mg/kg/day. The finding correlated with higher liver weights noted in treated females and was considered to be associated with the minimal follicular cell hypertrophy observed in the thyroid gland in some high dose animals.
In two males given 1000 mg/kg/day, minimal focal degeneration of the muscle layer was observed in the rectum. Due to the focal distribution and low severity of this finding, it was considered incidental and not related to test item administration.
Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test item.
Histopathological findings: neoplastic:
no effects observed

Effect levels

Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

open allclose all
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
endocrine system
Organ:
thyroid gland
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
Oral administration of the test item for 13 weeks in the rat was associated with an increase in liver weight in males and females at 250 and 1000 mg/kg/day and in kidney and thyroid gland weights in both sexes and at all dose levels.
At 1000 mg/kg/day, microscopic findings were observed in the liver of both sexes and thyroid glands in females. In the liver, there was arteritis/periarteritis in males, and minimal to mild centrilobular hepatocellular hypertrophy in both sexes. In the thyroid gland, there was minimal follicular cell hypertrophy in females.
At 250 mg/kg/day, microscopic findings were only seen in the liver. They consisted of arteritis/periarteritis in two males and minimal centrilobular hepatocellular hypertrophy in females.
Treatment-related findings at 50 mg/kg/day were confined to lower plasma phosphate levels and higher kidney weights in females only.
A dose level of 50 mg/kg/day was considered to be the No Observed Adverse Effect Level (NOAEL), as there were no treatment-related microscopic findings at this dose level.
Executive summary:

Groups of 10 male and 10 female Han Wistar rats were dosed by daily oral (gavage) administration of the test substance at a dose level of 50, 250 or 1000 mg/kg/day in 1% carboxymethylcellulose (aq), for a period of 13 weeks according to OECD 408 guideline.

The following parameters and end points were evaluated in this study from all animals: viability, clinical observations, body weights, food consumption and ophthalmoscopy examinations. A full functional observation battery (including motor activity) was performed for all animals during Week 12. Blood samples were collected from all animals during Week 13 for haematology, coagulation and clinical chemistry investigations and urine samples were collected for urinalysis during Week 13.

All animals were terminated after completion of at least 91 days of treatment and underwent a detailed necropsy examination with selected organs weighed. Tissues from all control animals and animals which received 1000 mg/kg/day were subjected to a comprehensive histological examination, with gross lesions and target tissues (where appropriate) examined from animals which received 50 and 250 mg/kg/day.

Formulation analysis revealed all samples were within acceptance criteria on all sampling occasions.

There were no unscheduled deaths, and there was no effect of treatment apparent on clinical observations, body weight, body weight gain or on food or water consumption.

Ophthalmoscopy evaluation did not reveal any treatment-related findings. Functional observation battery did not show an effect of treatment.

Clinical pathology investigations during Week 13 revealed increased fibrinogen in males and females at 1000 mg/kg/day. In addition, alanine and aspartate aminotransferase activities were increased in males given 1000 mg/kg/day. Total protein for both males and females of Groups 3 (250 mg/kg/day) and 4 (1000 mg/kg/day) and albumin and globulin for males and females of Group 4 (1000 mg/kg/day) were increased and sodium was increased, whilst phosphate was reduced for females at all dose levels.

Increases in liver and kidney weight in males and females at 250 and 1000 mg/kg/day, and an increase in kidney weights in females at 50 mg/kg/day were noted. Centrilobular hypertrophy consistent with the increased liver weights was observed in males and females given 1000 mg/kg/day and females at 250mg/kg/day. Thyroid hypertrophy was observed in the high dose females only. Arteritis/periarteritis was observed at 250 and 1000 mg/kg/day in male livers only.