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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-09-23 to 2009-06-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions
Cross-reference
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted 1998-09-21
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
480-070-0
EC Name:
-
Cas Number:
85-27-8
Molecular formula:
C14H14O2
IUPAC Name:
4-(1-phenylethyl)benzene-1,3-diol
Details on test material:
- Name of test material (as cited in study report): SymWhite® 377
- Synonym: SymWhite 377 (158281)
- Supplier: Symrise SA
- Physical state: white to beige powder
- Storage condition of test material: at room temperature and protected from light.
- Container: one opaque plastic container

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Sprague-Dawley, Crl CD® (SD) IGS BR, Caesarian Obtained, Barrier Sustained-Virus Antibody Free (COBS-VAF®)
- Source: Charles River Laboratories France, l’Arbresle, France.
- Age on the first day of treatment: 6 weeks old
- Weight on the first day of treatment: males: mean body weight of 197 g (range: 164 g to 220 g); females: mean body weight of 154 g (range: 131 g to 174 g)
- Housing: the animals were housed in a barriered rodent unit. The animals were housed in pairs, by sex and group, in suspended wire-mesh cages
(43.0 x 21.5 x 18.0 cm). A metal tray containing autoclaved sawdust (SICSA, Alfortville, France) was placed under each cage. Every 5 to 7 weeks, all the cages were moved.
- Diet (ad libitum): SSNIFF R/M-H pelleted maintenance diet (batch Nos. 1860828 and 7691203 (SSNIFF Spezialdiäten GmbH, Soest, Germany)), which was distributed weekly.
- Water (ad libitum): tap water (filtered with a 0.22 µm filter)
- Acclimation period: a period of 7 days before the beginning of the treatment period

On arrival, the animals were given a clinical examination to ensure that they were in good clinical condition.


ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Relative humidity: 50 ± 20%
- Ventilation: approximately 12 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: soybean oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS
- The test item was administered as a suspension in the vehicle. The test item was ground to a fine powder, using a mortar and pestle, and then mixed with the required quantity of vehicle in order to achieve the concentrations of 4, 12 and 40 mg/mL.
- The test item dosage forms were prepared at a frequency depending on their stability (up to 11 days as confirmed in CIT/Study No. 35284 AHS), stored at +4°C, protected from light, prior to use and delivered in brown flasks.

VEHICLE
- Batch no.: 067K0068
- Supplier: Sigma (Saint-Quentin-Fallavier, France)

ADMINISTRATION
- The quantity of dosage form administered to each animal was adjusted according to the most recently recorded body weight.
- A constant dosage-volume of 5 mL/kg/day was used.
- Control animals (group 1) received the vehicle alone.
- The dosage forms were stirred continuously throughout the dosing procedure.
- The dosage forms were left to come to room temperature before treatment and were administered at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The HPLC/UV analytical method for the determination of SymWhite® 377 in dosage form samples was provided by the Sponsor and this method was validated at the testing laboratory prior to dosage form analysis.The validation was based on the ICH Q6A guideline adopted in November 1999.

ANALYTICAL METHOD
PRINCIPLES OF ANALYTICAL METHOD
- Compound analyte: SymWhite 377

- Instrument/detector: High Performance Liquid Chromatography with Ultra-Violet detection at 280 nm (detector: dual λ absorbance detector 2487 (Waters), Series 1100 (Agilent Technologies))

- Preparation:
a. Control dosage forms (0 mg/mL): An aliquot (1 mL) of each control dosage form was sampled (by accurate weight) under magnetic stirring into a 200-mL volumetric flask and each flask was made to volume with dilution solvent before injection.
b. Test dosage forms (4, 12 and 40 mg/mL): an aliquot (1 mL) of each test item dosage form was sampled (by accurate weight) under magnetic stirring into a 200 mL volumetric flask and each flask was made to volume with dilution solvent. To achieve a target concentration of 10 μg/mL of SymWhite® 377, an additional dilution (2-fold, 6-fold and 20-fold for 4, 12 and 40 mg/mL, respectively) was carried out with dilution solvent before injection.
From the aliquot of dosage form sampled (weighed accurately), the real volume of the aliquot analyzed was determined (using the density of each dosage form) and therefore the value of the first dilution factor was calculated.

- Chromatographic conditions (HPLC/UV)
a. Pump: Series 1100 (Agilent Technologies), 2695 Alliance (Waters)
b. Mobile phase: Mobile phase A: 0.1% Formic acid in Milli-Q water, Mobile phase B: Acetonitrile
c. Gradient:
- 0 min, A: 70 %, B: 30 %
- 10 min, A: 30 %, B: 70 %
- 10.1 min, A: 0 %, B: 100 %
- 15 min, A: 0 %, B: 100 %
- 17 min, A: 70 %, B: 30 %
- 20 min, A: 70 %, B: 30 %
d. Column: YMC ODS-AQ, particle size = 5 μm, length = 150 mm, inner diameter = 3 mm
e. Flow rate: 0.6 mL/min
f. Temperature: 40°C
g. Oven: Series 1100 (Agilent Technologies), 2695 Alliance (Waters)
h. Detector: Dual λ absorbance detector 2487 (Waters), Series 1100 (Agilent Technologies)
i. Wavelength: 280 nm
j. Injector: Series 1100 (Agilent Technologies), 2695 Alliance (Waters)
k. Injected volume: 10 μL
l. Software: Empower 2 (Waters)
m. Retention time of test item: approx. 8.2 min
n. Analysis time: 20 min.

- Quantification: the concentration of the test item in administered dosage forms was determined from the mean response factor of SymWhite® 377 in standard solutions.
The dosage form concentrations were determined using the following:
[Concentration dosage form] = Area sample x Mean response factor X multiplication factor
Where:
Area sample = Area of sample
Mean response factor = Mean response factor of standard solution (n = 10)
Multiplication factor = dilution factor (also including conversion between units if required)

- Assay: diluted samples of dosage form were analyzed by High Performance Liquid Chromatography with Ultra-Violet detection.
One dilution was prepared for each sample and one injection was performed for each final dilution.
The test item peak area was determined for each sample and the corresponding concentration was calculated using the equation obtained from the calibration data. All the results are expressed as mg/mL of SymWhite® 377.

VALIDATION OF THE ANALYTICAL METHOD
The analytical procedure for determination of the SymWhite® 377 concentration was developed at the testing laboratory and was based on the analytical method provided by the Sponsor. The purpose of the study was to verify the following criteria: specificity, linearity, sensitivity, injection repeatability, accuracy and precision, and stability of standard and sample solutions. The solutions were prepared appropriately, and then analyzed by High Performance Liquid Chromatography with Ultra-Violet detection (HPLC-UV).
-Specificity: a vehicle sample, as well as 2 and 40 mg/mL formulation samples were prepared and analyzed. No interfering peaks were observed for the vehicle sample at the analyte retention time. No interfering peaks were observed for the formulation samples around the analyte retention time.
- Specificity in presence of degradation products: Samples of a 2 mg/mL dosage form in soybean oil were diluted in 0.1M HCl, 0.1MNaOH and 1% DMSO, stored for 24H at 50-60°C, and then analyzed by HPLC-DAD. No interfering peaks were observed under the main peak. One interfering peak partially co-eluted in the sample degraded by NaOH. As this peak is only partially co-eluting, it could be detected by HPLC-UV alone. Consequently, the method is still considered to be stability indicating.
- Linearity: standard solutions ranging from 1 to 15 μg/mL were prepared and analyzed.
The calibration curve was linear over a concentration range of from 1 μg/mL to 15 μg/mL (six concentration levels) as the obtained (r²) value was ≥ 0.99, the residuals of measured responses against response at nominal concentration were within ± 3%, and the intercept value was within ± 5% of response at nominal concentration.
- Accuracy and precision: working sample solutions made of vehicle, SymWhite® 377 and diluent were prepared, in triplicates, and at 80%, 100% and 120% of the nominal concentration. These samples were then analyzed.
Accuracy was demonstrated at each level, as the recovery was equal to or within 95-105% in each case.
Precision was demonstrated at each level, as the Coefficient of Variation (CV) was equal to or below 5% in each case.
- Injection repeatability: the repeatability of the assay method was tested with six successive injections of a standard solution at nominal concentration.
Injection repeatability was demonstrated as the CV was equal to or below 3%.
- Sensitivity: A standard solution at 0.1 μg/mL was prepared and analyzed. The obtained Signal-to-Noise (S/N) was superior to 10.
The control group dilution factor before injection is 200. Therefore, the absence of active material in a control group sample could be demonstrated at a level of 0.02 mg/mL.
- Stability in solutions: standard solutions and 2 and 40 mg/mL sample working solutions were analyzed on the day of dilution and after 24 hours and 4 days storage at room temperature, protected from light. Recovery was equal to or between 97-103% in each case.

Determination of dosage form homogeneity and stability:
Before the start of treatment the suitability of the proposed preparation process was confirmed by analysis of the homogeneity and stability. A range of dosage forms was prepared at levels which covered the lowest and highest concentrations proposed for use in the study, and then stored at +4°C and protected from light.
Duplicate samples were taken from three levels of the container (top, middle and bottom) on the day of preparation and on each sampling occasion. All samples were analyzed for test item content.
Samples taken just after preparation were analyzed immediately and the homogeneity/stability test was continued only when satisfactory initial results had been obtained.
Samples taken on each sampling occasion were analyzed immediately. On each occasion, the mean (n = 6) concentration was determined and compared to the nominal concentration, and the Coefficient of Variation (CV %) was calculated.
Acceptance criteria at each time-point:
- mean concentration = nominal concentration ± 15%,
- CV% < 10%.
The frequency of sampling was documented.

Determination of SymWhite®377 concentration in dosage forms:
The dosage form samples were assayed using a validated method, which was authorized by the Study Director prior to use in this study.
The test item concentration in samples of each control and test item dosage form prepared for use in weeks 1, 4, 8 and 13 was determined. Whenever possible these analyses were performed prior to administration of the dosage forms to the animals.
Acceptance criterion:
- measured concentration = nominal concentration ± 15%

RESULTS:
The test item concentrations in the administered dosage forms analyzed on the first and last day of treatment remained within an acceptable range of [-5.0% to +3.3%] of variation compared to the nominal values (please refer to "Any other information on materials and methods incl. tables" below for concentrations of SymWhite® 377 in administered dosage forms (table 1)) .
Duration of treatment / exposure:
at least 13 weeks (i.e. 92 days according to the necropsy schedule)
Frequency of treatment:
once a day, at approximately the same time
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (group 1), 20 (group 2), 60 (group 3) and 200 (group 4) mg/kg bw/day
Basis:
other: actual dose received
No. of animals per sex per dose:
10 males / 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: the dose-levels were selected in agreement with the Sponsor, following the results of previous studies:
- 7-day toxicity study (please refer to Section 7.5.1 Repeated dose toxicity: oral: k_Leuschner_2006) in which dose-levels of 300 and 1000 mg/kg/day elicited mortality and clinical signs of piloerection and hypoactivity. A decreased body weight was also observed,
- 4-week toxicity study (please refer to Section 7.5.1 Repeated dose toxicity: oral: k_Leuschner_2006) in which the dose-level of 200 mg/kg/day elicited a lower body weight gain and increased urea levels but no mortality or severe clinical signs.
Positive control:
no data

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
Time schedule:
- Mortality/morbidity: each animal was checked for mortality or signs of morbidity at least twice a day during the treatment period, including weekends and public holidays.
- General clinical observations: each animal was observed once a day, at approximately the same time, for the recording of clinical signs.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week until the end of the study

BODY WEIGHT: Yes
- Time schedule for examinations: once before group allocation, on the first day of treatment, and then once a week until the end of the study.

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes
- The quantity of food consumed by the animals of each cage was recorded once a week, over a 7-day period, during the study. Food consumption was calculated per animal and per day. If one of the two animals in the same cage died, the number of days for which that animal had been present in the cage was taken into consideration for the calculation of the food consumption.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- By visual appraisal of water bottles.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: on all animals, before the beginning of the treatment period and on animals of the control and high-dose groups (200 mg/kg/day dose group) on one occasion at the end of the treatment period (week 12).
- Examination: the pupils of the animals were dilated with tropicamide (Mydriaticum®, Laboratoires Théa, Clermont-Ferrand, France). After assessment of the corneal reflex (by instillation of tropicamide), the appendages, optic media and fundus were examined by indirect ophthalmoscopy (Oméga 180, Heine, Herrsching, Germany).

HAEMATOLOGY: Yes
Blood samples were taken from the orbital sinus of the animals (before the daily treatment) under isoflurane anesthesia, into tubes containing the appropriate anticoagulant. Prior to blood sampling, the animals were deprived of food for an overnight period of at least 14 hours, but they were not deprived of water. The following parameters were determined for all animals at the end of the treatment period and for prematurely sacrificed animals: erythrocytes, hemoglobin, mean cell volume, packed cell volume, mean cell hemoglobin concentration, mean cell hemoglobin, thrombocytes, leucocytes, differential white cell count with cell morphology (neutrophils, eosinophils, basophils, lymphocytes and large unstained cells and monocytes) and prothrombin time.

CLINICAL CHEMISTRY: Yes
Blood samples were taken from the orbital sinus of the animals (before the daily treatment) under isoflurane anesthesia, into tubes containing the appropriate anticoagulant. Prior to blood sampling, the animals were deprived of food for an overnight period of at least 14 hours, but they were not deprived of water. The following parameters were determined for all animals at the end of the treatment period and for prematurely sacrificed animals:sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea, creatinine, total bilirubin, total proteins, albumin, albumin/globulin ratio, total cholesterol, triglycerides, alkaline phosphatase, aspartate aminotransferase and alanine aminotransferase.

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the treatment period.
- Metabolism cages used for collection of urine: Yes, urine was collected in the presence of thymol crystals.
- Animals fasted: Yes, during urine collection, the animals were deprived of food for an overnight period of at least 14 hours, but they were not deprived of water.
- Parameters examined (for all animals): volume, pH, specific gravity, proteins, glucose, ketones, bilirubin, nitrites, blood (haemoglobin), urobilinogen, cytology of sediment (leucocytes, erythrocytes, cylinders, magnesium ammonium phosphate crystals, calcium phosphate crystals, calcium oxalate crystals and epithelial cells), appearance and colour.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Functional Observation Battery: detailed clinical observation, reactivity to manipulation or to different stimuli, motor activity.
- Time schedule for examinations: once after week 10.
Sacrifice and pathology:
On completion of the treatment period, after at least 14 hours fasting, all surviving animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination. Two males of the 200 mg/kg/day dose group were prematurely sacrificed in the same way.

GROSS PATHOLOGY: Yes
A complete macroscopic post-mortem examination was performed on all animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

ORGAN WEIGHTS:
The body weights were recorded for all animals before sacrifice at the end of the treatment period.
The following organs were weighed wet as soon as possible after dissection: adrenals, brain (including medulla/pons cerebellar and cerebral cortex), epididymides, heart, kidneys, liver, ovaries (including oviducts), spleen, testes, thymus and uterus (horns and cervix).
The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated. The ratio of organ weight to brain weight was also calculated because of the lower body weights of the test item treated groups.

HISTOPATHOLOGY: Yes
A microscopic examination was performed on all tissues listed below for animals of 0 mg/kg/day and 200 mg/kg/day dose groups and on the liver and kidneys of animals of the 20 mg/kg/day and 60 mg/kg/day dose groups.
Organs examined: macroscopic lesions, adrenals, aorta, brain (including medulla/pons cerebellar and cerebral cortex), cecum, colon, duodenum, epididymides, esophagus, heart, ileum with Peyer's patches, jejunum, kidneys, larynx, liver, lungs with bronchi, lymph nodes (mandibular and mesenteric), mammary glands/area, ovaries (including oviducts), pancreas, pituitary gland, prostate (dorso-lateral and ventral), rectum, salivary glands (sublingual and submandibular), sciatic nerve, seminal vesicles (including coagulation gland), skin, spinal cord (cervical, thoracic and lumbar), spleen, sternum with bone marrow, stomach with forestomach, testes, thymus, thyroids with parathyroids, trachea, urinary bladder, uterus (horns and cervix) and vagina.
Statistics:
Body weight, food consumption, hematology, blood biochemistry and urinalysis data:
Kolmogorov-Lilliefors test, Bartlett test, Fisher test, Dunn test, Mann-Whitney / Wilcoxon test and Dunnett test.
PathData software (version 6.2b5) was used for the statistical analysis of organ weight data (level of significance: 0.05 or 0.01):
Kolmogorov test, Bartlett test, F-test, Kruskal-Wallis, one-way analysis of variance, Dunn test, Wilcoxon test, Dunnett test and t-test

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
- Mortality: two males and one female treated at 200 mg/kg/day were found dead at the beginning of week 10 and two males treated at 200 mg/kg/day were prematurely sacrificed in week 13. Animals were in poor clinical condition, often showing body weight loss and clinical signs of hypoactivity,
hunched posture, piloerection and thin appearance. All these animals showed dosing-related inflammatory lesions of the respiratory tract and the female also showed marked cystitis and pyelonephritis. These deaths were considered to be related to the dosing procedure and not considered to be associated with the toxicity attributed to the test item.
There were no unscheduled deaths at the 0, 20 and 60 mg/kg/day dose levels.
-Clinical signs: the following clinical signs were observed at the 200 mg/kg/day dose level: hypersalivation (all animals), loud breathing (all animals), dyspnea (2/10 m; 1/10 f), abdominal breathing (3/10 m; 3/10 f), sneezing (1/10 f), thin appearance (3/10 m; 1/10 f), hunched posture (5/10 m; 5/10 f), piloerection (8/10 m; 3/10 f), pallor of extremities (1/10 m), hypoactivity (2/10 m), soiled urogenital region (2/10 m) and half-closed eyes (1/10 f).
At 200 mg/kg/day, hypersalivation was generally observed from week 1 throughout the study. Loud breathing tended to start in week 2 but also lasted throughout the study. Other clinical signs were observed during the second half of the dosing period; on one occasion or lasting for up to 5 weeks.
One female in each of the groups treated at 20 or 60 mg/kg/day showed hypersalivation, otherwise there were no treatment-related clinical signs at these dose-levels.

BODY WEIGHT AND WEIGHT GAIN
- Males:
Males treated at 200 mg/kg/day consistently had lower mean body weight gains than the controls, achieving statistical significance at regular intervals, and had mean body weight losses in weeks 9, 11 and 12. From weeks 1 to 9, the lower mean body weight gains were primarily caused by two or three of the same males with low body weights, however from week 9, mean body weights of this group were more than 10% lower than those of the controls and more males had lower body weights than the control range.
- Females:
Females treated at 200 mg/kg/day were less affected than the males, showing similar mean body weight gains to the controls throughout the study with the exception of week 1 where a statistically significantly lower mean body weight gain was recorded and weeks 9 and 12 when the females had a mean body weight loss.
- Over the treatment period as a whole (200 mg/kg/day dose level), both males and females had a markedly lower mean body weight gain.
- There were no effects of treatment with the test item on the mean body weight gain of animals treated at 20 or 60 mg/kg/day. Females treated at 20 mg/kg/day did have a moderately lower overall mean body weight gain but in the absence of an effect at 60 mg/kg/day or in the males treated at 20 mg/kg/day, this was considered to be unrelated to treatment with the test item.

FOOD CONSUMPTION AND WATER CONSUMPTION
- Males:
Males treated at 200 mg/kg/day generally had higher food consumption than the controls from week 2 until the end of the study, achieving statistical significance on occasion.
Males treated at 60 mg/kg/day tended to have slightly higher food consumption than the controls for the first five weeks of treatment after which values were similar to the controls.
-Females:
Females treated at 200 mg/kg/day also showed a tendency towards higher food consumption than the controls during weeks 2 to 8.
Females treated at 60 mg/kg/day did not have noticeably higher mean food consumption than the controls.

- Higher water consumption was also observed, necessitating more frequent filling of water bottles than usual (twice or three times per week instead of once), for animals treated at 200 mg/kg/day.
- There were no effects of treatment with the test item on food consumption of the males or females treated at 20 mg/kg/day

OPHTHALMOSCOPIC EXAMINATION
No abnormalities were observed in any animal.

HAEMATOLOGY
- Overall white blood cell counts were similar to the controls or marginally increased, however neutrophil count was statistically significantly higher in males and females treated at 200 mg/kg/day and monocyte count was statistically significantly higher in the same males. Some degree of hemoconcentration was observed in males and females treated at 200 mg/kg/day (statistically significantly increased red blood cell counts, hemoglobin levels and hematocrit).
- Prothrombin time was longer in males treated at all dose-levels, statistically significantly so at 60 or 200 mg/kg/day, but given the complete lack of dose-relationship, this effect is considered unlikely to be related to treatment with the test item.
(Please refer to table 1 in "Any other information on results incl. table" below for results on haematology)

CLINICAL CHEMISTRY
- Plasma electrolyte concentrations were affected in both males and females treated at 60 or 200 mg/kg/day; increased sodium concentration in males and females treated at 200 mg/kg/day and decreased potassium and chloride concentrations in males and females treated at 60 or 200 mg/kg/day. Differences were dose-related and observed in both sexes and were therefore considered to be related to treatment with the test item.
- Urea concentration was statistically significantly increased in males at all dose-levels and in females treated at 60 or 200 mg/kg/day. In conjunction, creatinine concentration was statistically significantly increased in males and females treated at 200 mg/kg/day. These effects are consistent between sexes and dose-related and are considered to be related to treatment with the test item.
- Liver enzyme alanine aminotransferase activity was statistically significantly increased in males and females treated at 200 mg/kg/day, when compared with the controls. The effect was minimal in females and moderate in males and was considered to be related to treatment with the test item.
- Cholesterol and triglycerides concentrations were statistically significantly increased in females treated at 60 or 200 mg/kg/day and glucose concentration was statistically significantly decreased in females treated at 200 mg/kg/day. Males treated at the same dose-levels showed minimal, non-statistically significant differences to controls in these parameters. An effect of treatment cannot be excluded.
- Males treated at 200 mg/kg/day showed a statistically significant increase in inorganic phosphorus concentration, when compared with the controls, which was not observed in females also treated at 200 mg/kg/day. An effect of treatment cannot be excluded.
(Please refer to table 2 in "Any other information on results incl. table" below for results on clinical chemistry)

URINALYSIS
- Volume produced by the males and females treated at 200 mg/kg/day was statistically significantly increased when compared with the controls (explained by the higher water consumption) and consequently the specific gravity was statistically significantly decreased. There was also a slight effect on the specific gravity of the urine produced by the males treated at 60 mg/kg/day.
- Males and females treated at 60 or 200 mg/kg/day showed higher incidences of nitrites in the urine and presence of calcium phosphate crystals.
- None of the males treated at 200 mg/kg/day and only one out of ten males treated at 60 mg/kg/day had detectable protein in the urine, compared to nine out of ten of the control males having detectable levels. Half of the males treated at 20 mg/kg/day also had undetectable levels of protein in the urine. These differences are probably actually related to the higher urine volume and more dilute urine rather than a decrease in urinary proteins.
(Please refer to table 3 in "Any other information on results incl. table" below for results on urinalysis)

NEUROBEHAVIOUR
- Motor activity: males treated at 200 mg/kg/day made a lower mean number of movements during the 60-minute session than the control animals, most noticeable for the number of rearing movements.
- Females treated at the same dose-level made a higher number of movements than the controls so this effect in the males is probably related to the lower body weight gain over the study and poorer general condition of the animals.
- No effects of treatment on mean number of movements at 20 or 60 mg/kg/day.

ORGAN WEIGHTS
- Organ weight changes reaching statistical significance and considered to be test item-related were recorded in the kidneys of both males (absolute: -20%; p<0.01)(relative: +46%; p<0.01) and females (relative: +27%; p<0.01) that received 200 mg/kg/day. The spectrum of histopathological findings observed in the kidneys (i.e. tubular basophilia, simple/cystic tubular dilatation, single cell necrosis, tubular casts, etc.) of this dose group were considered to be the morphological correlates to this organ weight change.

GROSS PATHOLOGY
No macroscopic findings were observed that were considered to distinguish test item-treated rats from rats that received the vehicle only.

HISTOPATHOLOGY: NON-NEOPLASTIC
- A spectrum of degenerative/regenerative changes was observed in the kidneys of a few rats that received 60 mg/kg/day, and all males and most females that received 200 mg/kg/day of the test item. These changes included an increased incidence and/or severity of multifocal to widespread renal tubular basophilia, an increased incidence and/or severity (slight to moderate) of multifocal to widespread renal tubular dilatation (simple and/or cystic), that involved cortical/medullary/papillary tubules, minimal to slight increased apoptosis and single cell degeneration/necrosis of renal tubular epithelium (i.e. cortex/medulla), minimal increase in mitoses of tubular epithelium and suppurative pyelonephritis, with associated tubular necrosis and/or fibrosis. In addition, minimal to severe thymic atrophy was observed in two controls and in eight animals treated at 200 mg/kg/day. Minimal to marked lymphoid atrophy was observed in the spleen from three males treated at 200 mg/kg/day.
- No toxic effects were observed at the hsitopathological evaluation at 20 or 60 mg/kg/day in males and 20 mg/kg/day in females.

Effect levels

Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1

Sex

Dose-level (mg/kg/day

Male

Female

0

20

60

200

0

20

60

200

White blood cells (G/L)

9.97

9.09

12.41

11.19

7.27

6.12

7.11

7.36

Neutrophils

1.18

1.35

1.50

3.00**

0.80

0.81

0.74

1.71**

Monocytes

0.18

0.18

0.24

0.38**

0.15

0.12

0.15

0.19

Red blood cells (T/L)

8.75

8.50

8.51

9.48*

7.62

7.64

7.86

8.64**

Hemoglobin (g/dL)

15.8

15.6

15.6

17.1**

14.8

14.7

15.0

16.7**

Hematocrit (L/L)

0.45

0.44

0.44

0.48*

0.41

0.41

0.42

0.46**

Prothrombin time (s)

16.5

17.3

17.5*

17.2**

16.6

16.6

16.2

16.3

Statistically significant: *: p<0.05, **: p<0.01

Table 2

Sex

Dose-level (mg/kg/day

Male

Female

0

20

60

200

0

20

60

200

Sodium (mmol/L)

143.3

143.5

143.4

145.2*

142.4

142.0

141.8

143.1

Potassium (mmol/L)

4.25

4.11

3.58**

3.37**

3.72

3.83

3.42

3.26*

Chloride (mmol/L)

105.0

105.0

100.6**

97.1**

105.0

104.8

101.0**

100.3**

Urea (mmol/L)

2.9

3.6*

4.6**

7.9**

4.3

4.6

5.5*

8.3**

Creatinine (μmol/L)

47

47

48

58**

53

54

53

65*

ALAT (IU/L)

43

37

42

77**

30

34

32

42**

Glucose (mmol/L)

7.12

7.05

6.83

6.51

6.96

6.98

6.82

6.06*

Cholesterol (mmol/L)

1.1

1.2

1.3

1.4

1.3

1.6

1.7*

1.7*

Triglycerides (mmol/L)

0.48

0.47

0.76

0.66

0.28

0.33

0.37*

0.50**

In. phosphorus (mmol/L)

1.98

1.92

1.88

2.24**

1.68

1.67

1.64

1.73

Statistically significant: *: p<0.05, **: p<0.01; In. : inorganic

Table 3

Sex

Dose-level (mg/kg/day

N

Male

Female

0

20

60

200

0

20

60

200

10

10

10

8

10

10

10

9

Volume (mL)

7

8

11

27**

9

5

12

24**

Specific gravity

1041

1035

1030*

1014

1033

1041

1028

1013**

The following parameters are presented by number of animals affected:

Nitritesa

0

0

3

7

1

1

2

5

Calcium phosphate crystalsa

- none observed

10

9

8

1

7

4

3

2

- few in some fields

 

 

2

1

1

1

3

5

- few in all fields

 

1

 

6

2

4

3

2

- several in all fields

 

 

 

 

 

1

1

 

Proteinsa

- negative

1

5

9

8

7

7

10

9

- ~0.3 g/L

5

3

1

 

2

3

 

 

- ~1 g/L

4

2

 

 

1

 

 

 

Applicant's summary and conclusion

Conclusions:
At the dose-level of 200 mg/kg/day, mean body weight gain of males and females was markedly lower than that of the controls; males in fact showed a mean body weight loss in week 9 and then from week 11, females in weeks 9, 12 and 13, although food consumption of both sexes was consistently higher.
Loud breathing, dyspnea and hypersalivation were observed in many animals and males had a decreased number of movements in the motor activity assessment. Water consumption was remarked as being higher (increased frequency of water bottle filling) and urinary volume was significantly increased and contained nitrites and calcium phosphate crystals at higher incidences than in the controls. Animals nevertheless showed hemoconcentration (increased red blood cell count, hemoglobin concentration and hematocrit), and increased neutrophil and monocyte counts. Electrolytes showed differences to controls in both males and females (sodium was higher and potassium and chloride were lower) and increases in urea, creatinine and alanine aminotransferase were observed. Cholesterol and triglycerides were also higher in females and glucose concentration was lower, and inorganic phosphorus was higher in males.
Kidney weight was statistically significantly higher in both males and females and microscopic findings (degenerative/regenerative changes) were observed in all males and females. Thymic atrophy and spleenic lymphoid atrophy were also noted.

There were no notable in-life effects at 60 mg/kg/day; no mortality, no differences in mean body weight gains or hematology parameters and only one female showed hypersalivation. However, potassium and chloride were decreased and both males and females had increased urea concentration. Females also had increased cholesterol and triglycerides concentrations. Nitrites were present in the urine at a higher incidence than in the controls and more animals had calcium
phosphate crystals than in the control group. Five males and two females showed microscopic findings (degenerative/regenerative changes) in the kidneys.

At 20 mg/kg/day, no in-life effects were recorded other than one female with hypersalivation and an increased urea concentration in males. No microscopic findings were observed at a higher incidence or severity than in the control animals.

Under the experimental conditions of this study, the NOAEL was considered to be 20 mg/kg/day and the target organ was established to be the kidney.