5-fluoro-2-methoxypyrimidin-4(3H)-one

Endpoint summary

Description of key information

Oral

Under the experimental conditions of this study and based on the histopathological findings, a No Observed Adverse Effect Level (NOAEL) is considered to be 15 mg/kg/day for both males and females.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 September 2005 to 20 October 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.43 (Neurotoxicity Study in Rodents)

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA 799, 9620-62-158

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Sprague-Dawley rat, Rj Han: SD, Indenm of Organism Pathogen Specific Han (IOPS Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females (if applicable) nulliparous and non-pregnant: Not specified
- Age at study initiation: Approximately 6 weeks old
- Weight at study initiation: Males: mean weight 221 g (207 to 234 g); females: mean weight 174 g (159 to 188 g)
- Fasting period before study: Not required. Animals fasted for at least 14 hours before terminal sacrifice.
- Housing: The animals were individually housed in suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm). A metal tray, containing autoclaved sawdust was placed under each cage.
- Diet: ad libitum
- Water: ad libitum access to bottles containing tap water (filtered with a 0.22 μm filter)
- Acclimation period: 6 days before the beginning of the treatment period

DETAILS OF FOOD AND WATER QUALITY
The batch of diet and sawdust were analysed by the suppliers for composition and contaminant levels. Bacterial and chemical analyses of water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (pesticides, heavy metals and nitrosamines). No contaminants were present in the diet, drinking water or sawdust at levels which could be expected to interfere with, or prejudice, the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 50 ± 20 % (relative)
- Air changes: Approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod: 12 h/ 12 h light/dark cycle (07:00 - 19:00)

IN-LIFE DATES
From: 15 September 2005
To: 20 October 2005

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Remarks:

0.5 % aqueous methylcellulose solution

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS
The vehicle was degassed by sonication for at least 30 minutes and then kept under nitrogen atmosphere until saturation of the dead volume. The test material was administered as a suspension in the vehicle. The test material was ground to fine powder using a mortar and pestle, suspended in the vehicle in order to achieve the concentrations of 3, 10 and 30 mg/mL and then homogenised using a magnetic stirrer.
The quantity of dosage form administered to each animal was adjusted according to the most recently recorded body weight. The dosage forms were stirred continuously throughout the dosing procedure.

DOSE VOLUME: A constant dosage-volume of 5 mL/kg/day was used

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

HOMOGENEITY
Two dosage forms were prepared at 3 and 200 mg/mL of test material to cover all the concentrations intended for use in this study. From each dosage form, duplicate samples were taken at three different levels of the container (top, middle, bottom) and analysed for concentration of the test material to evaluate the homogeneity.
The results of the analyses demonstrated the homogeneity of each dosage form analysed (3 and 200 mg/mL) just after preparation (under nitrogen atmosphere). Furthermore, there was a satisfactory correspondence between the nominal and the measured concentrations of the test material in the vehicle.

STABILITY
Each dosage form prepared for homogeneity analysis was sampled after 0 (just after preparation, homogeneity test), 4 and 9 days of storage at +4 °C (under nitrogen atmosphere). The aliquots sampled on day 4 were stored frozen at -20 °C pending analysis on last sampling occasion (day 9). The mean concentration measured on day 0 (homogeneity) was taken as the initial value for the stability test.
The results of the analyses demonstrated the satisfactory stability of the two dosage forms investigated (3 and 200 mg/mL) over a 9-day period at +4 °C (under nitrogen atmosphere).

CONCENTRATION
The concentration of samples taken from each dosage form (including the control) prepared for use in weeks 1 and 4 was determined.
An aliquot of each dosage form was diluted appropriately then analysed by High Performance Liquid Chromatography with Ultra-Violet detection at 220 nm. The concentration of the test material was determined from a calibration curve of peak area against concentration of the test material in standard solutions (external standard calibration).
The analytical method used was validated prior to analysis of the study samples.

- Sample preparation
An aliquot (1 mL) of each test dosage form under magnetic stirring was sampled (weighed accurately) in a test tube and diluted 10-fold with mobile phase or in a 100-mL volumetric flask and each flask was made to volume with mobile phase. To achieve target concentrations in a range 0.1 to 10 μg/mL, serial dilutions (100 to 200-fold) were carried out with mobile phase depending on the initial concentration. The control dosage form was analysed without dilution.
From the aliquot of dosage form sampled (weighed accurately), the real volume of the aliquot analysed was determined (taking into account the density of each dosage form) and the value of the first dilution factor was calculated.

- Chromatographic conditions (HPLC/UV)
Pump: 114M (Beckman), Series 1100 (Agilent Technologies)
Mobile phase de-gassed: acetonitrile 32 V; water 68 V; triethylamine 0.02 V; orthophosphoric acid 0.05 V
Column: Zorbax RX-C8 (Agilent Technologies), 250 x 4.6 mm, particle size = 5 μm
Flow rate: 1.5 mL/min
Temperature: 30 °C
Oven: Sup RS Stabitherm, Series 1100 (Agilent Technologies)
Detector: Dual λ absorbance detector 2487 (Waters), Series 1100 (Agilent Technologies)
Wavelength: 200 nm
Injector: Autosampler 717plus (Waters), Series 1100 (Agilent Technologies)
Injected volume: 10 μL
Data acquisition software: Multichrom 2 (Fisons Instruments)
Retention time of test material: approx. 2 mins
Analysis time: 5 mins

- Calibration curve
Peak areas were determined for standard solutions ranging from 0.1 to 10 μg/mL (six levels). A calibration curve was obtained by linear regression analysis of peak areas against concentrations. The regression analysis of the calibration data gave an equation of the following form: Y = aX + b
Where
Y = peak area of test material (μV.s)
X = concentration of test material (mg/mL)
a = slope value
b = intercept

- Assay
Diluted samples of dosage form were analysed 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 material peak area was determined for each sample and the corresponding concentration was calculated using the equation obtained from the calibration data.

- Validation of the analytical method
The specificity, limit of quantification, linearity, repeatability of injections, accuracy and precision (Coefficient of Variation: CV %) of the analytical method were determined.
The specificity of the analytical method was demonstrated as follows: analysis of a standard solution of the test material in mobile phase and analysis of 0.5 % aqueous methylcellulose (vehicle) without dilution. No relevant interference was observed on chromatograms between the test material and the vehicle.
The limit of quantification of the analytical method was established as 0.1 μg/mL for a standard solution of the test material. This limit corresponds to a limit of quantification of 0.0001 mg/mL for the test material in 0.5 % aqueous methylcellulose.
Linearity was checked by analysis of three different sets of six standard solutions containing 0.1, 0.5, 1, 2, 5 and 10 mg/L of test material in mobile phase. Satisfactory linearity was demonstrated in the range 0.1 to 10 mg/L since the coefficients of determination obtained were higher than 0.999.
For the repeatability of injections, replicate analysis (n = 10) of a solution containing 2.64 mg/L of the test material gave satisfactory results since the coefficients of variation obtained were as follows: 2 % based on peak height and 1 % based on peak area. Based on these results, the repeatability of injections of the analytical method was validated taking into account peak area.
Accuracy and precision were determined. Six analyses of dosage forms containing 3 and 200 mg/mL of the test material in 0.5 % aqueous methylcellulose were carried out. Samples were diluted appropriately with the mobile phase before analysis.
The analytical method was validated and considered to be suitable for the analysis of the samples of the study.

A satisfactory agreement was observed between the nominal and actual concentrations of the test material in the administered dosage forms since the deviations from nominal concentration were in an acceptable range of ± 10 %.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

7 days/week (once daily)

Dose / conc.:

15 mg/kg bw/day (actual dose received)

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

150 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose-levels were selected in agreement with the Sponsor, on the basis of the results of a 7-day range-finding toxicity study by oral route performed in the same species in which the test material was administered for 7 days to Sprague-Dawley rats at the dose-levels of 0, 30, 150 and 300 mg/kg. In this study, at the dose-level of 30 mg/kg, neither treatment-related clinical signs nor relevant clinical pathological findings were noted, but the body weight gain was lower in females. At the dose-level of 150 mg/kg, round back and piloerection was noted in one male. Body weight loss was noted in the females and lower body weight gain was noted in the males given 150 mg/kg. No relevant clinical pathological findings were noted at this dose-level. At the dose-level of 300 mg/kg, all the animals were found dead or sacrificed prematurely, generally without post-mortem macroscopic findings.
- Rationale for animal assignment: During the acclimation period, the required number of animals (20 males and 20 females) were selected according to body weight and clinical condition. They were then allocated to groups (by sex), according to a computerised stratification procedure, so that the average body weight of each group was similar.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each animal was checked for mortality or signs of morbidity at least twice a day during the treatment period, including weekends and public holidays. Any animal showing signs of poor clinical condition, especially if death appears imminent, were humanely killed. Each animal was observed at least once a day, at approximately the same time, for the recording of clinical signs.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made on all animals outside the home cage, in a standard arena, once before the beginning of the treatment period and then once a week until the end of the study. Observations included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded at least once before group allocation, on the first day of treatment and then once a week until the end of the study.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: The quantity of food consumed by the animals of each cage were recorded once a week until the end of the study.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No, although eyes were examined as part of the FOB

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period (day 28), blood samples were taken from the orbital sinus of the animals (before the daily treatment) and collected into tubes containing the appropriate anticoagulant (EDTA for haematology parameters, sodium citrate for PT).
- Anaesthetic used for blood collection: Yes, under isoflurane anaesthesia
- Animals fasted: Yes. Prior to blood sampling, the animals were deprived of food, but not water, for an overnight period of at least 14 hours.
- How many animals: All animals
- Parameters examined: Erythrocytes (RBC), Haemoglobin (HB), Mean cell volume (MCV), Packed cell volume (PCV), Mean cell haemoglobin concentration (MCHC), Mean cell haemoglobin (MCH), Thrombocytes (PLAT), Leucocytes (WBC), Differential white cell count with cell morphology (neutrophils (N), eosinophils (E), basophils (B), lymphocytes (L), monocytes (M)) and Prothrombin time (PT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period (day 28), blood samples were taken from the orbital sinus of the animals (before the daily treatment) and collected into tubes containing the appropriate anticoagulant (lithium heparin).
- Anaesthetic used for blood collection: Yes, under isoflurane anaesthesia
- Animals fasted: Yes. Prior to blood sampling, the animals were deprived of food, but not water, for an overnight period of at least 14 hours.
- How many animals: All animals
- Parameters examined: Sodium (Na+), Potassium (K+), Chloride (Cl-), Calcium (Ca++), Inorganic phosphorus (I.PHOS), Glucose (GLUC), Urea (UREA), Creatinine (CREAT), Total bilirubin (TOT.BIL.), Total proteins (PROT), Albumin (ALB), Albumin/globulin ratio (A/G), Total cholesterol (CHOL), Triglycerides (TRIG), Alkaline phosphatase (ALP), Aspartate aminotransferase (ASAT) and Alanine aminotransferase (ALAT).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Animals were evaluated once at the end of the treatment period.
- Dose groups that were examined: All animals
- Battery of functions tested: The evaluation included a detailed clinical examination, measurement of reactivity to manipulation or to different stimuli and motor activity. The animals were randomized. All animals were observed in the cage, in the hand and in the standard arena. The following parameters were assessed and graded:
- "touch escape" or ease of removal from the cage,
- in the hand: fur appearance, salivation, lachrymation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis),
- in the standard arena (2-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypy, behaviour, breathing, ataxia and hypotonia.
Then, the following parameter measurements, reflexes and responses were recorded: touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex, landing foot splay and rectal temperature (at the end of observation).
Finally, motor activity of all animals was measured once by automated infra-red sensor equipment over a 60-minute period.

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
On completion of the treatment period, after at least 14 hours of fasting, all surviving animals were asphyxiated by carbon dioxide and sacrificed by exsanguination.
The body weights of all animals sacrificed at the end of the treatment period were recorded before sacrifice, and the organs specified were weighed wet as soon as possible after dissection. The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated. The organs weighed were: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus and thyroids with parathyroids.
A complete macroscopic post-mortem examination was performed on all study animals, including those that dies or were sacrificed in extremis. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

HISTOPATHOLOGY: Yes
For all study animals, the tissues specified were preserved in 10 % buffered formalin (except for the eyes and Harderian glands which were fixed in Davidson's fixative, and the testes and epididymides which were preserved in Bouin's fluid). All tissues required for microscopic examination were embedded in paraffin wax, sectioned at approximately four microns in thickness and stained with haematoxylin-eosin.
Microscopic examination was performed on the following tissues for all animals of the control and high dose groups (Groups 1 and 4) sacrificed at the end of the treatment period and for all animals that died prematurely: adrenals, brain (including medulla/pons cerebellar and cerebral cortex), cecum, colon, duodenum, epididymides, heart, ileum, jejunum, kidneys, liver, lungs with bronchi, lymph nodes (mandibular and mesenteric), ovaries with oviducts, prostate, sciatic nerve, seminal vesicles, 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), vagina and all macroscopic lesions.
Microscopic examination was performed on the following tissues for all animals of the low- and intermediate-dose groups (Groups 2 and 3) sacrificed on completion of the treatment period: heart, liver, kidneys, spleen, lungs and all macroscopic lesions.

Statistics:

Statistical analyses of body weight, food consumption, haematology, blood biochemistry and organ weight data were carried out using the following statistical procedures: Kolmogorov-Lilliefor’s test, logarithmic transformation, assessment of homogeneity of variances between groups, Bartlett’s test, Fisher’s test, Dunn’s test, Mann-Whitney/Wilcoxon’s test, Student’s test and Dunnett’s test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Slight to moderate tremors were observed in all females given 150 mg/kg/day of the test material on days 2 to 3 or 14 to 16. At 150 mg/kg/day, staggering gait was observed in one female (days 15 and 16) and two males (from day 27) from the same group. These signs were considered to be related to the treatment with the test material as limited to the top dose group only.
The other clinical signs observed (i.e., scabs, neck abscess, hindlimb increased size, chromodacryorrhea) were noted with a low-incidence, on a few days only and are commonly noted in animals of this strain and age. Thus, they were considered to be without relationship to the treatment with the test material.
No clinical signs were observed at 15 and 50 mg/kg/day.

Mortality:

mortality observed, treatment-related

Description (incidence):

One female from the high dose-level group was found dead on day 29 of the study (last day of treatment). Slight tremors were observed in this animal on day 2, and no longer thereafter. No clinical signs were observed prior to death. At microscopic examinations, the following observations were noted: focal myocarditis, minimal periportal fat vacuolation and slight hypertrophy of centrilobular hepatocytes in the liver, foci of granulomatous pneumonitis and perivascular inflammatory cell infiltration in lungs, ovaries and thymus congestion. As most of these findings were noted in animals at scheduled sacrifice, this death could be related to the treatment with the test material. No deaths were noted in the other groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight gain was lower in treated animals than in control animals during all the treatment period. Although not statistically significant (except for the last week of treatment in males), this effect was dose-related and thus considered to be related to the treatment with the test material. At the end of the treatment period, a dose-related reduced mean body weight was noted in all treated groups (-4, -6 and -11 % in males treated with 15, 50 and 150 mg/kg/day of the test material respectively, and -6 and -9 % in females at 50 and 150 mg/kg/day, respectively), reaching a statistically significant level (p<0.05) on day 29 for the males at 150 mg/kg/day.

The mean final body weight of males given 150 mg/kg/day was significantly lower than in controls (p<0.05). Although the mean final body weight of females given the highest dose was lower than controls, the difference was not statistically significant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The food consumption was not affected by the treatment with the test material at any of the dose-levels tested in this study.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The prothrombin time was significantly higher in animals given 150 mg/kg/day of the test material than in controls (+9 % in males and+ 10 % in females, p<0.05). This could be related to the treatment with the test material.
Statistically significantly higher platelets count was noted at 150 mg/kg/day in females (+25 %). As this effect was not dose-related and the values were within the historical data range, it was probably not related to the treatment with the test material. No relevant modifications of haematological parameters were noted at 15 and 50 mg/kg/day.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The major differences between control and treated animals are presented in Table 1.
At the end of the treatment period, the following changes in blood biochemical parameters were noted in the intermediate and high-dose groups:
- Higher urea levels both in males and females at 150 mg/kg/day (+73 and +88 %, respectively) and in females (+38 %) at 50 mg/kg/day.
- Higher creatinine levels in all treated groups except in females at 15 mg/kg/day (+14 % in males and + 18 % in females from the high-dose group).
- Lower glucose levels in females at 150 mg/kg/day (-31 %)
- Lower triglyceride levels in males at 150 mg/kg/day (-57 %) and at 50 mg/kg/day (-42 %).
- Male and female animals from the high-dose group exhibited lower alkaline phosphatase activity (-23 and -28 %, respectively).
- Higher aspartate aminotransferase activity was observed in all animals from the high-dose group (+123 % in males and +58 % in females).
As they were dose-related, these effects were considered to be related to the treatment with the test material.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Staggering gait was reported in two males given 150 mg/kg/day of the test material. As it was only observed in the high dose-level group, it was considered to be related to the treatment with the test material.
No abnormal observations were noted during the FOB examinations at 15 and 50 mg/kg/day. No abnormal reactions to stimuli were noted at any of the dose-levels tested. Higher mean horizontal movements were observed at 50 mg/kg/day in males when compared to controls (609.0 ± 276.5 vs. 523.8 ± 112.3 units), this was particularly due to one animal and was not dose-related. Thus it was not considered to be related to the treatment with the test material.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The relevant differences in organ weights recorded are reported in Table 2.
The mean relative weight of the liver was statistically significantly greater in both sexes given 150 mg/kg/day than in controls (p<0.05 for males, p<0.01 for females). This difference was correlated with the microscopic finding of centrilobular hypertrophy.
The mean relative weight of the heart was statistically significantly greater in males given 150 mg/kg/day than in controls (p<0.01). This inter-group difference may have resulted from an increase in myocarditis among males given the highest dose. No similar organ weight change was noted in females in which a similar but less severe increase in myocarditis was seen at microscopy.
The significantly greater mean relative weights of the epididymides and testes in males given 150 mg/kg/day than in controls (p<0.01 and p<0.05 respectively) were, in the absence of any correlating macroscopic or microscopic findings, considered to have arisen by chance or as a result of the significant difference in final body weight.
Similarly, since no microscopic counterpart could be seen for the decreased absolute weight of the thymus in females given 150 mg/kg/day (p<0.05) this finding was also considered to have arisen fortuitously.
There were no other significant inter-group differences in organ weight.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no macroscopic findings that could be related to the treatment with the test material.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The daily oral administration of the test material to rats at a dose-level of 150 mg/kg/day for 4 weeks produced treatment-related pathological changes in the heart, kidneys, liver and spleen of both sexes and in the lungs of males.

- Heart: When compared to controls, there was a greater incidence and severity of focal myocarditis in both sexes given 150 mg/kg/day and in males given 50 mg/kg/day. 1/5 females given 50 mg/kg/day had minimal focal myocarditis. This single incidence may have arisen spontaneously but since there was no myocarditis present in controls or in females given 15 mg/kg/day an effect of treatment cannot be discounted. The myocarditis was morphologically indistinguishable from lesions of so-called "murine progressive cardiomyopathy" and consisted of single or multiple foci of mononuclear inflammatory cells and degenerate cardiac myofibres. The distribution of the lesion included the atria and ventricles and in males the cardiac muscle surrounding the pulmonary vessels. The effect was greater in males than in females.

- Kidneys: Minimal to slight acute tubular necrosis, characterised by multifocal, coagulative-type necrosis of contiguous cells within cross sections of the cortical tubules, was present in 1/5 males and 3/5 females given 150 mg/kg/day. No inflammatory response was associated with the necrosis. There was a greater incidence and severity of foci of basophilic (regenerating) tubules in males from the high-dose group than in controls. There were no changes that could be related to the treatment with the test material in animals given 15 or 50 mg/kg/day.

- Liver: Minimal to slight hypertrophy of centrilobular hepatocytes was present in all the animals given 150 mg/kg/day. No hypertrophy was observed in any of the other animals on study.

- Lungs: In males given 150 mg/kg/day, there was minimal to slight myositis of the cardiac muscle surrounding the pulmonary veins. This finding was not observed in females or in males from the other treated groups.

- Spleen: Minimal extramedullary haematopoiesis was observed in all males and 4/5 females from the control group but in only 1/5 males given 150 mg/kg/day and in none of the females from this dose group. The incidence and severity of this finding in animals given 15 or 50 mg/kg/day were comparable to controls.
All of the other pathological findings encountered were considered to have arisen spontaneously.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg bw/day (actual dose received)

System:

cardiovascular

Organ:

heart

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Table 1: Summary of Selected Blood Biochemical Parameters

Parameter	Dose level (mg/kg/day)
	0	15	50	150
Males
Urea (mmol/L)	3.7	4.4	4.7	6.4**
Creatinine(μmol/L)	49	53**	55**	56**
Glucose (mmol/L)	6.27	5.81	5.83	5.43
Triglyceride (lU/L)	1.18	0.97	0.68**	0.51**
ALP (lU/L)	548	629	547	423*
ASAT (lU/L)	93	89	106	207**
Females
Urea (mmol/L)	4.8	4.9	6.6*	9.0**
Creatinine(μmol/L)	51	53	59**	60**
Glucose (mmol/L)	7.01	5.74	5.53	4.85*
Triglyceride (lU/L)	0.33	0.35	0.25	0.36
ALP (lU/L)	431	391	348	311*
ASAT (lU/L)	86	114	111	136**

Statistically significantly different from controls; *p < 0.05 and **p < 0.01

 

Table 2: Summary of Selected Organ Weights

Organ (and Sex)	Parameter	Dose level (mg/kg/day)
		0	150
Liver (males)	absolute (g)	12.85	13.32
	relative (% body weight)	3.13	3.69*
Liver (females)	absolute (g)	6.81	8.59
	relative (% body weight)	3.12	4.39**
Heart (males)	absolute (g)	1.35	1.40
	relative (% body weight)	0.33090	0.38681**
Testes (males)	absolute (g)	3.34	3.32
	relative (% body weight)	0.81759	0.91996*
Epididymides (males)	absolute (g)	0.96880	1.02
	relative (% body weight)	0.23717	0.28315**
Thymus (females)	absolute (g)	0.54960	0.41450*
	relative (% body weight)	0.25309	0.21155

Statistically significantly different from controls; *p < 0.05 and **p < 0.01

Conclusions:

Under the experimental conditions of this study and based on the histopathological findings, a No Observed Adverse Effect Level (NOAEL) is considered to be 15 mg/kg/day for both males and females.

Executive summary:

The repeated dose toxicity of the test material was investigated via the oral route in accordance with the standardised guidelines EU Method B.7, EU Method B.43 and US EPA 799 under GLP conditions.

A total of 20 male and 20 female Sprague-Dawley rats were randomly allocated to three treated groups and one control group. The treated groups received the test material by gavage at the dose-levels of 15, 50 or 150 mg/kg/day for 4 weeks. The control group received the vehicle only (0.5 % aqueous methylcellulose) under the same experimental conditions. The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded weekly. Haematological and blood biochemical investigations and a Functional Observation Battery (FOB) were performed at the end of the treatment period. On completion of the treatment period, all surviving animals were killed and submitted to a full macroscopic post-mortem examination. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on selected tissues from animals in the control and all treated groups.

One female from the high-dose group was found dead on day 29 of the study (last day of treatment). No clinical signs were observed prior to death. As the histopathological findings in this animal were also noted in animals at scheduled sacrifice, this death was considered to be related to the treatment with the test material. No deaths were noted in the other groups.

Slight to moderate tremors were observed in all females given 150 mg/kg/day of the test material on a few days at the beginning or at mid-dosing period. Staggering gait was observed in one female (on days 15 and 16) and two males (from day 27) of the same group. No clinical signs were observed at 15 and 50 mg/kg/day.

Except for staggering gait reported in two males given 150 mg/kg/day of the test material, no abnormal observations were noted during the FOB examinations. No abnormal reactions to stimuli were noted at any of the dose-levels tested in the study. When compared to controls, no relevant modifications of motor activity were noted in treated groups.

The body weight gain was lower in treated animals than in control animals during the dosing period, and this in a dose-related manner. This effect was very marked during the last week of treatment in males given 150 mg/kg/day (+1 g vs. +28 g in controls, p<0.01) and a mean body weight loss was noted in females of the same group (-1 g vs. +4 g in controls). At the end of the treatment period, a tendency to a dose-related lower mean body weight was noted in all treated groups when compared to controls (-4, -6 and -11 % in males treated with 15, 50 and 150 mg/kg/day of the test material respectively, and -6 and -9 % in females at 50 and 150 mg/kg/day, respectively), reaching a statistically significant level (p<0.05) only in males at 150 mg/kg/day).

No relevant modifications in food consumption were noted in treated animals when compared to control animals.

Except for the prothrombin time that was significantly higher in animals given 150 mg/kg/day of the test material than in controls, no relevant modifications of haematological parameters were noted in treated animals.

At the end of the treatment period, and when compared to controls, higher level of creatinine was noted in all treated animals, except in females at 15 mg/kg/day. Higher urea level was noted at 150 mg/kg/day both in males and females, and at 50 mg/kg/day in females only. A lower glucose level was observed in females from the high dose-level group, and a lower triglyceride level was noted in males from the intermediate and high dose-level groups. Alkaline phosphatase activity was significantly lower in animals from the high dose-level group and aspartate aminotransferase activity was significantly higher in animals from the same group.

Higher mean relative weight of the liver was noted in both sexes and higher relative weight of the heart was noted in males only at 150 mg/kg/day.

No macroscopic findings that could be related to the treatment with the test material were observed. When compared to the control group, a higher incidence and severity of focal myocarditis was observed in both sexes given 150 mg/kg/day. This effect was greater in males than in females. It was also observed in 4/5 males and 1/5 females given 50 mg/kg/day. A minimal to slight acute kidney tubules necrosis was present in both sexes given 150 mg/kg/day, with a greater incidence in females (3/5 versus 1/5 animals). In males given 150 mg/kg/day, there was a greater incidence and severity of foci of basophilic (regenerating) tubules than in controls. Liver lesions (hypertrophy of centrilobular hepatocytes) were present in all the animals given 150 mg/kg/day. A minimal to slight myositis of the cardiac muscle surrounding the pulmonary veins was observed in males only from the high-dose group in lungs.

Under the experimental conditions of this study and based on the histopathological findings, a No Observed Adverse Effect Level (NOAEL) is considered to be 15 mg/kg/day for both males and females .

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

15 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

A single study conducted in accordance with standardised guidelines and under GLP conditions is available. The quality of the database is therefore considered to be acceptable for risk assessment.

System:

cardiovascular

Organ:

heart

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral

The repeated dose toxicity of the test material was investigated via the oral route in accordance with the standardised guidelines EU Method B.7, EU Method B.43 and US EPA 799 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

A total of 20 male and 20 female Sprague-Dawley rats were randomly allocated to three treated groups and one control group. The treated groups received the test material by gavage at the dose-levels of 15, 50 or 150 mg/kg/day for 4 weeks. The control group received the vehicle only (0.5 % aqueous methylcellulose) under the same experimental conditions. The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded weekly. Haematological and blood biochemical investigations and a Functional Observation Battery (FOB) were performed at the end of the treatment period. On completion of the treatment period, all surviving animals were killed and submitted to a full macroscopic post-mortem examination. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on selected tissues from animals in the control and all treated groups.

One female from the high-dose group was found dead on day 29 of the study (last day of treatment). No clinical signs were observed prior to death. As the histopathological findings in this animal were also noted in animals at scheduled sacrifice, this death was considered to be related to the treatment with the test material. No deaths were noted in the other groups.

Slight to moderate tremors were observed in all females given 150 mg/kg/day of the test material on a few days at the beginning or at mid-dosing period. Staggering gait was observed in one female (on days 15 and 16) and two males (from day 27) of the same group. No clinical signs were observed at 15 and 50 mg/kg/day.

Except for staggering gait reported in two males given 150 mg/kg/day of the test material, no abnormal observations were noted during the FOB examinations. No abnormal reactions to stimuli were noted at any of the dose-levels tested in the study. When compared to controls, no relevant modifications of motor activity were noted in treated groups.

The body weight gain was lower in treated animals than in control animals during the dosing period, and this in a dose-related manner. This effect was very marked during the last week of treatment in males given 150 mg/kg/day (+1 g vs. +28 g in controls, p<0.01) and a mean body weight loss was noted in females of the same group (-1 g vs. +4 g in controls). At the end of the treatment period, a tendency to a dose-related lower mean body weight was noted in all treated groups when compared to controls (-4, -6 and -11 % in males treated with 15, 50 and 150 mg/kg/day of the test material respectively, and -6 and -9 % in females at 50 and 150 mg/kg/day, respectively), reaching a statistically significant level (p<0.05) only in males at 150 mg/kg/day).

No relevant modifications in food consumption were noted in treated animals when compared to control animals.

Except for the prothrombin time that was significantly higher in animals given 150 mg/kg/day of the test material than in controls, no relevant modifications of haematological parameters were noted in treated animals.

At the end of the treatment period, and when compared to controls, higher level of creatinine was noted in all treated animals, except in females at 15 mg/kg/day. Higher urea level was noted at 150 mg/kg/day both in males and females, and at 50 mg/kg/day in females only. A lower glucose level was observed in females from the high dose-level group, and a lower triglyceride level was noted in males from the intermediate and high dose-level groups. Alkaline phosphatase activity was significantly lower in animals from the high dose-level group and aspartate aminotransferase activity was significantly higher in animals from the same group.

Higher mean relative weight of the liver was noted in both sexes and higher relative weight of the heart was noted in males only at 150 mg/kg/day.

No macroscopic findings that could be related to the treatment with the test material were observed. When compared to the control group, a higher incidence and severity of focal myocarditis was observed in both sexes given 150 mg/kg/day. This effect was greater in males than in females. It was also observed in 4/5 males and 1/5 females given 50 mg/kg/day. A minimal to slight acute kidney tubules necrosis was present in both sexes given 150 mg/kg/day, with a greater incidence in females (3/5 versus 1/5 animals). In males given 150 mg/kg/day, there was a greater incidence and severity of foci of basophilic (regenerating) tubules than in controls. Liver lesions (hypertrophy of centrilobular hepatocytes) were present in all the animals given 150 mg/kg/day. A minimal to slight myositis of the cardiac muscle surrounding the pulmonary veins was observed in males only from the high-dose group in lungs.

Under the experimental conditions of this study and based on the histopathological findings, a No Observed Adverse Effect Level (NOAEL) is considered to be 15 mg/kg/day for both males and females .

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance requires classification with respect to repeated dose toxicity via the oral route as STOT RE Category 2 (H373: May cause damage to organs).