Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 June - 01 July 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: United States Environmental Protection Agency (EPA). Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, July 2000.
Deviations:
no
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: bulk
Details on test material:
- Name of test material (as cited in study report): Anhydrothymidine
- Substance type: White powder
- Physical state: powder
- Analytical purity: 99.7%
- Purity test date: not stated
- Lot/batch No.: 073904/05
- Expiration date of the lot/batch: 11 December 2008
- Stability under test conditions: stable
- Storage condition of test material: At room temperature in the dark

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 6 weeks.
- Weight at study initiation: around 160 g males, 135 g females
- Fasting period before study:
- Housing: Group housing of 5 animals per sex in Macrolon cages (MIV type, height 18 cm; during overnight activity monitoring individual housing in MIII type; height 15 cm.) with sterilized sawdust as bedding material and paper as cage-enrichment. No cage-enrichment was provided during overnight activity monitoring.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet
- Water (e.g. ad libitum): Free access to tap water
- Acclimation period: At least 5 days before the start of treatment under laboratory conditions


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5 – 22.3
- Humidity (%): 31 - 89
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Oral gavage, using a plastic feeding tube.
Formulations were placed on a magnetic stirrer during dosing.
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing, and were homogenized to visually acceptable levels. No correction was made for the purity of the test substance.


VEHICLE
- Water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of formulations were analyzed on a single occasion during the in-life phase for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours was also determined (highest and lowest concentration).
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily, 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
vehicle
No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
- Dose selection rationale: based on the pilot experiment
- Rationale for animal assignment (if not random): random
Positive control:
not required

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality / Viability


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily


BODY WEIGHT: Yes
- Time schedule for examinations: Weekly.


FOOD CONSUMPTION: Weekly.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination at the end of the treatment
- Anaesthetic used for blood collection: Yes, iso-flurane anaesthesia
- Animals fasted: Yes, overnight (with a maximum of 20 hours) before blood sampling
- How many animals: all
- Parameters examined:
White blood cells
Differential leucocyte count
neutrophils, lymphocytes, monocytes,
eosinophils, basophils
Red blood cells
Reticulocytes
Red blood cell distribution width
Haemoglobin
Haematocrit
Mean corpuscular volume
Mean corpuscular haemoglobin
Mean corpuscular haemoglobin concentration
Platelets
Prothrombin time
Activated Partial thromboplastin time


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination at the end of the treatment
- Animals fasted: Yes, overnight (with a maximum of 20 hours) before blood sampling
- How many animals: all
- Parameters examined:
Alanine aminotransferase
Aspartate aminotransferase
Alkaline phosphatase
Total Protein
Albumin
Total Bilirubin
Urea
Creatinine
Glucose
Cholesterol
Sodium
Potassium
Chloride
Calcium
Inorganic Phosphate


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 4 of treatment
- Dose groups that were examined: all
- Battery of functions tested: motor activity, hearing ability, pupillary reflex, static righting reflex and grip strength
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. Tissues/organs mentioned in parentheses were not examined by the pathologist since there were no changes in macroscopic appearance indicative of (potential) toxicity.
Identification marks: not processed
Adrenal glands
(Aorta)
Brain [cerebellum, mid-brain, cortex]
Caecum
Cervix
(Clitoral gland)
Colon
Duodenum
Epididymides
(Eyes with optic nerve [if detectable] and
Harderian gland)
(Female mammary gland area)
(Femur including joint)
Heart
Ileum
Jejunum
Kidneys
(Larynx)
(Lacrimal gland, exorbital)
Liver
Lung, infused with formalin
Lymph nodes - mandibular, mesenteric
(Nasopharynx)
(Oesophagus)
Ovaries
(Pancreas)
Peyer's patches [jejunum, ileum] if detectable
(Pituitary gland)
(Preputial gland)
Prostate gland
Rectum
(Salivary glands - mandibular, sublingual)
Sciatic nerve
Seminal vesicles
(Skeletal muscle)
(Skin)
Spinal cord -cervical, midthoracic, lumbar
Spleen
Sternum with bone marrow
Stomach
Testes
Thymus
Thyroid including parathyroid [if detectable]
(Tongue)
Trachea
Urinary bladder
Uterus
Vagina
All gross lesions

ORGAN WEIGHTS:
Adrenal glands
Brain
Epididymides
Heart
Kidneys
Liver
Spleen
Testes
Thymus


HISTOPATHOLOGY: Yes
The following slides were examined by a pathologist:
- all tissues collected at the scheduled sacrifice from all Main group 1 and 4 animals,
- all gross lesions.

All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
Statistics:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test1 (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test2 (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test3 was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicity were noted during the observation period.
Incidental findings such as alopecia (in the neck or on the right cheek), scabs (in the neck) and brown staining were noted in individual animals. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of treated animals remained in the same range as controls over the study period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after allowance for body weight was similar between treated and control animals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.
Haematological findings:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes occurred in haematological parameters of treated rats.
Statistically significant changes were noted in females only. Slightly decreased monocyte percentage was noted at 1000 mg/kg/day and increased red blood cell distribution width (RDW) was noted at 50 mg/kg/day. These findings were considered to be of no toxicological significance as they remained within the range considered normal for rats of this age and strain and occurred in the absence of a clear treatment-related distribution.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Cholesterol was decreased in males in a dose related distribution. Statistical significance was noted at 1000 mg/kg/day only.
A slight increase of sodium level was noted in females at 50 and 150 mg/kg/day. This finding was considered to be of no toxicological significance as it occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.
The variation in motor activity did not indicate a relation with treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No toxicologically significant changes were noted in organ weights and organ to body weight ratios.
Absolute and relative spleen weights of females at 50 and 150 mg/kg/day were noted to be increased. These findings in females occurred as a result of a slightly lower control value and in the absence of a dose related distribution. A decreased relative spleen weight of males at 1000
mg/kg/day was noted, which was well within the range considered normal for rats of this age and strain. No corroborative findings were noted in macroscopic or microscopic examination.
Therefore changes in spleen weight were considered not to be toxicologically significant. Other finding was a decreased absolute brain weight in females at 150 mg/kg/day. This finding occurred in the absence of a dose related distribution and remained within the range considered
normal for rats of this age and strain. Therefore this finding was considered to be not toxicological relevant.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Necropsy did not reveal any toxicologically relevant alterations.
Incidental findings were considered to be of no toxicological significance since they occurred in
the absence of a treatment-related distribution, and are occasionally seen among rats used in
these types of studies. These findings included, nodules in the liver, epididymides, diaphragm
and lungs, pelvic dilation, scab formation, growing together of lungs with aorta, pale
discolouration of lungs and fluid in uterus.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
There were no microscopic findings recorded which could be attributed to treatment with the test substance.
All microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.
Details on results:
MORTALITY
No mortality occurred during the study period.

CLINICAL SIGNS
No clinical signs of toxicity were noted during the observation period.
Incidental findings such as alopecia (in the neck or on the right cheek), scabs (in the neck) and brown staining were noted in individual animals. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance.


BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain of treated animals remained in the same range as controls over the study period.

FOOD CONSUMPTION
Food consumption before or after allowance for body weight was similar between treated and control animals.


HAEMATOLOGY
No toxicologically relevant changes occurred in haematological parameters of treated rats.
Statistically significant changes were noted in females only. Slightly decreased monocyte percentage was noted at 1000 mg/kg/day and increased red blood cell distribution width (RDW) was noted at 50 mg/kg/day. These findings were considered to be of no toxicological significance as they remained within the range considered normal for rats of this age and strain and occurred in the absence of a clear treatment-related distribution.


CLINICAL CHEMISTRY
Cholesterol was decreased in males in a dose related distribution. Statistical significance was noted at 1000 mg/kg/day only.
A slight increase of sodium level was noted in females at 50 and 150 mg/kg/day. This finding was considered to be of no toxicological significance as it occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain


NEUROBEHAVIOUR
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.
The variation in motor activity did not indicate a relation with treatment.


ORGAN WEIGHTS
No toxicologically significant changes were noted in organ weights and organ to body weight ratios.
Absolute and relative spleen weights of females at 50 and 150 mg/kg/day were noted to be increased. These findings in females occurred as a result of a slightly lower control value and in the absence of a dose related distribution. A decreased relative spleen weight of males at 1000 mg/kg/day was noted, which was well within the range considered normal for rats of this age and strain. No corroborative findings were noted in macroscopic or microscopic examination. Therefore changes in spleen weight were considered not to be toxicologically significant.
Other finding was a decreased absolute brain weight in females at 150 mg/kg/day. This finding occurred in the absence of a dose related distribution and remained within the range considered normal for rats of this age and strain. Therefore this finding was considered to be not toxicological relevant


GROSS PATHOLOGY
Necropsy did not reveal any toxicologically relevant alterations.
Incidental findings were considered to be of no toxicological significance since they occurred in the absence of a treatment-related distribution, and are occasionally seen among rats used in these types of studies. These findings included, nodules in the liver, epididymides, diaphragm and lungs, pelvic dilation, scab formation, growing together of lungs with aorta, pale discolouration of lungs and fluid in uterus.

HISTOPATHOLOGY
There were no microscopic findings recorded which could be attributed to treatment with the test substance.
All microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects

Target system / organ toxicity

Key result
Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Wistar rats were treated with the test substance for 28 consecutive days by daily oral gavage at dose levels up to 1000 mg/kg/day.
In clinical biochemistry measurements cholesterol was statistically significant decreased in males at 1000 mg/kg/day. Since the cholesterol levels were within the range considered normal for rats of this age and strain and since no corroborative findings were noted in microscopic examination, this finding was considered not toxicological relevant.
No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, macroscopic examination, organ weights and microscopic examination).
From the results presented in this report a definitive No Observed Adverse Effect Level (NOAEL) for the test substance of 1000 mg/kg/day was established.