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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:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: EK1008F023
- Expiration date of the lot/batch: 09 augustus 2018
- Purity test date:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity:
- Specific activity:
- Locations of the label:
- Expiration date of radiochemical substance:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS:

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Wistar Hannover (HsdRCCHanTM.WIST)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS rl
- Females (if applicable) nulliparous and non-pregnant: [yes/ ]
- Age at study initiation:
- Weight at study initiation: 85-107 gram
- Fasting period before study:no
- Housing: up to 3 /sex/ cage (polysulfone solid bottomed)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 21 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/- 2 °C
- Humidity (%): 55 +/- 15%
- Air changes (per hr): 15-20 / hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The test item was administered orally by gavage at a dose volume of 5 mL/kg body weight. Control animals received the vehicle alone at the same dose volume.
The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal
Vehicle:
CMC (carboxymethyl cellulose)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 1% Methocel ®A4C in softened by reverse osmosis water.
- Amount of vehicle (if gavage): 5ml/kgbw
- Lot/batch no. (if required):
- Purity:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the formulation prepared once during pre-treatment period and during Weeks 1 and 13 of the study were analysed to check the homogeneity and concentration.
Duration of treatment / exposure:
All animals were dosed once a day, 7 days a week, for a minimum of 13 consecutive weeks followed by a recovery period of 4 weeks for 5 males and 5 females from groups 1 and 4. Animals in the main phase were dosed up until the day before necropsy.
Frequency of treatment:
All animals were dosed once a day, 7 days a week
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 /sex for groups 10 and 30 mg/kgbw/day
15/sex for control and 100mg/kgbw/day
Control animals:
yes

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
- Cage side observations checked in table [No.?] were included. before treatment + time interval post treatment (5-30 min / 1.5 -2 hours)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before treatment + once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekley

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): weekly/ cage
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

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: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / Not specified
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once during 13 week treatment
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to necropsy
- Anaesthetic used for blood collection: Yes (identity) isofluorane
- Animals fasted: Yes (overnight befor necropsy)
- How many animals:
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to necropsy
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [No.?] were examined.

URINALYSIS: No
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / Not specified
- Animals fasted: Yes / No / Not specified
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / landing footplay

IMMUNOLOGY: No
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Statistics:
Standard deviations were calculated as considered appropriate. For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If the data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied.
The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off. Statistical analysis of histopathological finding was carried out by means of a nonparametric Kolmogorov-Smirnov test.
Sperm analysis : Standard deviations were calculated as considered appropriate. For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If the data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied.
The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off. Statistical analysis of histopathological finding was carried out by means of a nonparametric Kolmogorov-Smirnov test.
sperm analysis: Statistical analysis: Kruskall Wallis test + William’s test if group differences are different from control at p<0.05

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Piloerection and hairloss were occasionally observed in some male and female animals, mainly at the high dose levels, during the treatment period. These signs were no longer observed in male animals during the recovery period.
No changes of toxicologically relevance were found at the weekly clinical examination, which included an evaluation of neurotoxicity during treatment and recovery periods.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two cases of premature death occurred during the study.
One control male was found dead during the study (Day 61). Decreased activity, pallor and piloerection were observed in this animal from 2 days before death. The cause of death of was due to the injury of the urogenital tract.
A single female rat from Group 2 was sacrificed for humane reasons during the treatment period (Day 52). Decreased activity, damaged and impaired right hindlimb were seen in this animal prior to humane sacrifice. The relevant macroscopic finding was the rupture of right hindlimb and forelimb confirmed at histopathology as bone fracture, probably due to an incidental trauma.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):

Slight but statistically significant decreases in body weight (from -6% to -10%) were observed in high and mid-dose female animals from Day 29 of the treatment period and in the high dose males from Day 85. These decreases were still observed during the recovery period in animals of both sexes.
Slight but statistically significant reductions in absolute body weight gain were also observed in males of Group 4, starting from Day 15 up to the end of the treatment period (ranging from -12% to -16%) and in females, where a more pronounced effect was noticed, starting from Days 15, 29 and 36 (in Groups 4, 3 and 2, respectively) to Day 92 of the treatment period (ranging from -12% to -31%).
These decreases gradually disappeared during the recovery period and were not considered as adverse.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
No relevant changes were observed in food consumption in male animals, during the treatment and recovery period.
Very slight but statistically significant decreases, ranging from -6% to -15%, were observed in female animals from all treated groups (with a slight dose-relation), starting from Days 8 (Groups 3 and 4) and 29 (Group 2).
These decreases were no longer observed during the recovery period, and were not considered advers.
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Animals with no ocular abnormality were selected for the study by an ophthalmoscopic examination performed before the start of treatment. All animals were re-examined in Week 13 of treatment (Day 87) and no treatment-related findings were detected.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Dosing Phase
Statistically significant fluctuations of some haematological parameters were recorded in some treated males, such as: decrease of mean corpuscular haemoglobin concentration in animals dosed at 10 mg/kg/day (3%), increase of the same parameter in those receiving 100 mg/kg/day (1%), increase of reticulocytes in males dosed at 10 mg/kg/day and decrease of lymphocytes in those treated at 100 mg/kg/day (25%). The female dosed at 100 mg/kg/day (animal no. 81) had lymphocytes higher than controls (38% above mean control data).
Due to the absence of dose-relation, the slight severity and/or the inconsistency between sexes, the above changes were considered unrelated to treatment.
Recovery Phase
Compared with controls, statistically significant increases of mean corpuscular volume (6%) and reticulocytes (21%) were recorded in males dosed at 100 mg/kg/day.
These findings were not observed during the dosing phase, therefore they were considered unrelated to treatment.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Dosing Phase
Compared with controls, females dosed at 100 mg/kg/day showed decrease of creatinine (1%) and albumin (8%). Albumin was also decreased in females dosed at 10 and 30 mg/kg/day (8% and 10%, respectively). Due to the slight severity, these changes were considered of no toxicological relevance.
Other statistically significant differences were recorded between controls and animals dosed at 10 and/or 30 mg/kg/day, such as: decrease of albumin/globulin ratio in animals of both sexes, decreases of aspartate aminotransferase, chloride and calcium in females. Since these changes were not dose-related, they were considered unrelated to treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Dosing phase
Slight but statistically significant reduction in rearing was noted in treated animals towards the end of treatment. The rearing was reduced in males on Days 45 and 76, while females showed statistically significant reduction in rearing on Days 42, 49, 63, 70 and 90 of the treatment period. Reduction in urinalysis, of no toxicologically relevance, was also noticed in males on Day 90.
Since these findings were sporadic and not consistent, they were considered to be incidental fluctuations not related to treatment.
No other differences were noted at the weekly observations of animals at removal from the cage and in an open arena.
Recovery phase
A slight increase in rearing observed in high dose females on Day 14 (+18%) was considered to be incidental.
No other relevant findings were found at the weekly observations of animals at removal from the cage and in an open arena during the recovery period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No changes of toxicological significance were observed on terminal body weight, absolute and relative organ weights in the main and recovery phase animals.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes were noted at post mortem in the main and recovery phase animals. All macroscopic changes were related to physiological/agonal changes or were considered to be incidental.
Neuropathological findings:
no effects observed
Description (incidence and severity):
No relevant differences that could be considered treatment-related were observed at func- tional tests (sensory reactivity, landing footsplay, grip strength) performed at the end of treatment and recovery periods.
Motor activity measurements performed at the end of the treatment and recovery periods did not show any toxicologically significant differences between treated animals and controls.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Final sacrifice
Treatment-related findings were seen in the liver of males and females receiving Tellurium Dioxide at 10 mg/kg/day.
The hepatic changes consisted in a vacuolation as micro and macrovesicular fatty change like (steatosis). Morphologically, in the macrovesicular vacuolation, the hepatocytes con- tained a large, single vacuole which fills the cytoplasm and displaces the nucleus to the periphery, while the hepatocytes with microvesicular steatosis contained many small lipid droplets in the cytoplasm. Such hepatocellular injury was seen associated with an increased inflammation characterized by the presence of inflammatory aggregates, i.e. cell clusters containing different types of immune cells such as lymphocytes and macrophages, in males and females receiving Tellurium Dioxide at 100 mg/kg/day (high dose group) and with an increased severity in high dose females.
The remaining lesions reported were considered spontaneous and/or incidental pathology, known to occur spontaneously in untreated Sprague Dawley rats of this age under our experimental conditions.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.
Recovery sacrifice
The histopathological evaluation of the liver did not show any relevant changes in high dose animals, sacrificed after 4 weeks of recovery period, when compared with controls.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No differences were observed at sperm analysis including sperm motility and concentration, and cauda weight between the control and the high dose group at the end of treatment.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: For the different evaluation parameters effects were registrated, however the results were or; not dose related and considered incidental ; or they were slight and recovered.

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, slight signs of treatment-related effects (clinical signs, reductions in body weight and food consumption) were observed during the in vivo phase of the study, mainly at the high dose level of 100 mg/kg bw/day and, with lower extent at the mid-dose level of 30 mg/kg bw/day. At microscopic examination, changes were observed at the end of treatment period in the liver of animals dosed at 10 mg/kg bw/day. These changes were no longer present at the end of recovery period. Although the changes were observed at all the dose levels investigated with a dose-related trend, they were completely reversible and, as such, they were not considered to be adverse.
Therefore, it can be concluded that the No Observed Adverse Effect Level (NOAEL) for this study is 100 mg/kg bw/day.