Registration Dossier

Administrative data

Description of key information

The main effect observed upon oral exposure in the different sub-acute and sub-chronic available studies is reduction of weight and weight gain. At microscopic examination also changes in the liver were noted. However the severeness of the effects vary within the different studies and affected animals recover upon cessation of the treatment.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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
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
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:
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:
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
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
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
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.
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.
Critical effects observed:
no
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.
Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
see justification for read-across section 13.1
Reason / purpose:
read-across source
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
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:
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:
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
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
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
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.
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.
Critical effects observed:
no
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.
Endpoint:
repeated dose toxicity: oral
Remarks:
other: dose range finder (7days)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2012-09-18 to 2012-09-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
no guideline required
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633, from SPF colony
- Age at study initiation: Young adult rats, less than 9 weeks old
- Weight at study initiation:Phase 1: Males: 263-270 g Females: 192-196 g; Phase 2: Males: 231-260 g; Females: 176-215 g
- Fasting period before study: no data
- Housing: Standard laboratory conditions during the study, Type II and III polycarbonate cage, up to 5 animals of the same sex and group/cage.
- Diet (e.g. ad libitum): ssniff® SM R/M-Z+H ad libitum
- Water (e.g. ad libitum): tap water from municipal supply ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.7 – 23.7 °C
- Humidity (%): 31- 63 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): light 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Route of administration:
oral: gavage
Vehicle:
other: 1% aqueous Methylcellulose
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): 1% aqueous Methylcellulose (abbreviation: 1% MC) was used as vehicle.
- Concentration in vehicle:
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of tellurium dioxide dosing formulations for concentration and homogeneity was performed at the Test Site. Top, middle and bottom duplicate samples were taken from test item formulations once during Phase 2, one set to analyze which was collected in replicates and one set as a back-up, if required for any confirmatory analyses. Similarly, one sample was taken in duplicate from the Group 1 (control) solution for concentration measurements. The concentration of tellurium was analysed using ICP/AES.

The achieved concentrations were in the range of 96.7 and 102.8%. No test item was identified in the control samples.
Duration of treatment / exposure:
7 days
Frequency of treatment:
7 days/ week
Remarks:
Doses / Concentrations:
0 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
62.5 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
250 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5 per sex/ dose
Control animals:
yes, concurrent vehicle
Details on study design:
Phase 1 animals were treated by single dose oral gavage administration followed by a 7 day observation period and necropsy. The starting dose level (1000 mg/kg bw) was set by the Sponsor in consultation with the Study Director, based on available data and information from previous experimental work, including the results of an acute oral toxicity study.
Following the administration of 1000 mg/kg dose, all animals survived and no overt sign of toxicity was observed. Based on the results the Phase 2 started at 3 dose levels of 62.5, 250 and 1000 mg/kg bw/day.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Clinical observations were made twice daily, before and after treatment.
Observation was performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behaviour pattern, changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations: on Day 0 and at least on Days 3, 6 and 7 (prior to necropsy, fasted if scheduled euthanasia).

FOOD CONSUMPTION AND COMPOUND INTAKE: yes

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 AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on Day 7, blood samples for clinical pathology evaluation (haematology, coagulation, and clinical biochemistry) were collected immediately prior to the scheduled necropsy, by cardiac puncture under pentobarbital anaesthesia.
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes, After an overnight period of food deprivation of animals
- How many animals: all
- Parameters checked in table below were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on Day 7, blood samples for clinical pathology evaluation (haematology, coagulation, and clinical biochemistry) were collected immediately prior to the scheduled necropsy, by cardiac puncture under pentobarbital anaesthesia.
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table below were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
At the end of Phase 2 on Day 7, blood samples for clinical pathology evaluation (haematology, coagulation, and clinical biochemistry) were collected immediately prior to the scheduled necropsy, by cardiac puncture under pentobarbital anaesthesia.
After an overnight period of food deprivation of animals, 3 blood samples were collected, for haematology (approximately 1.2 mL blood in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), for blood clotting times (approximately 1.4 mL blood for APTT and PT measurements, in tubes with sodium citrate as anticoagulant) and one to obtain serum (approximately 1 mL blood as practical in tubes with no anticoagulant) for clinical chemistry.

On Day 7, all animals were euthanised under pentobarbital anaesthesia by exsanguination and subjected to gross necropsy.
After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.


No histopathology evaluation was performed.
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):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study. No clinical signs were observed during the study. All animals were symptom-free.
Dark faeces were observed in all animals at 250 and 1000 mg/kg/day on Day 6.

BODY WEIGHT AND WEIGHT GAIN
Body weight gain suppression was observed in both sexes at 1000 mg/kg/day, resulting in terminal body weight lower by approximately 4% (males) and 8% (females), compared to the control mean. In 4 of 5 females, negligible body weight gain or slight body weight loss were observed.
There were no toxicologically significant or adverse effects observed on the body weight following the administration of the test item at doses of 62.5 and 250 mg/kg/day for 7 consecutive days.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Lower food consumption was recorded for both males and females at 1000 mg/kg/day during the entire treatment period. Compared to the control mean, the mean values were lower by approximately 8% in males and 28% in females.
There were no adverse effects on the animal food consumption or differences ascribed to test item administration at doses of 62.5 and 250 mg/kg/day.

HAEMATOLOGY
Compared to controls, mean relative reticulocyte count was lower in both males and females at 1000 mg/kg/day. The differences were statistically significant (p<0.01). All values were within the normal range.
In females at 1000 mg/kg/day mean platelet count was significantly lower, when compared to the control mean. Lower platelet count was found in 4 of 5 females. (Table 2). Slightly lower mean platelet volume (MPV) was also measured for High dose females, however, individual values were not proportional to the individual platelet count.
Compared to control means, slightly higher relative monocyte count was measured in females treated at 62.5 mg/kg/day. The difference attained statistical significance (p<0.01).

CLINICAL CHEMISTRY
No test item related adverse effects at any dose level were noted in clinical chemistry parameters. All parameters were within the normal physiological ranges for the age, strain and sex of rats on test.

ORGAN WEIGHTS
The mean absolute and relative thymus weights adjusted for both brain and terminal body weight were significantly lower than the controls in females treated at 1000 mg/kg/day. The decrease was in the range of 33% for absolute value and 29-35% for relative values and were statistically significant (p<0.05 for absolute and body weigh relative values, p<0.01 for brain relative values)
Higher than control liver weights were noted at 1000 mg/kg/day in females. The increase was approximately of 13% for body weight relative weights and attained statistical significance (p<0.05).
Compared to the control mean, the absolute and relative weights of kidneys were lower in males at 1000 mg/kg/day by approximately 16% (absolute values) and the difference were statistically significant (p<0.05 for absolute and, p<0.01 for relative values). Lower relative weights of kidneys were also noted in the Low and Mid dosed males (p<0.05).

GROSS PATHOLOGY
There was no evidence of test item-related macroscopic findings in animals at necropsy following a 7-day administration of the test item, with the exception of black discoloration of the digestive content of stomach and intestines noted in males at 250 and 1000 mg/kg/day and in all treated groups of females.

Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: 7 days exposure
Critical effects observed:
not specified
Conclusions:
The Maximum Tolerated Dose (MTD) was found to be below 1000 mg/kg bw/day.
No adverse effect was observed at 250 mg/kg/day.
Executive summary:

Administration of tellurium dioxide to Wistar rats by oral gavage, daily for 7 days, at a dose level of 1000 mg/kg bw/day was associated with body weight gain suppression in both sexes, mild effect in clinical chemistry parameters (lower relative reticulocyte counts in both sexes, lower platelet counts and higher serum calcium concentration in females), an lower kidney weights in males and lower thymus weight in females. No adverse effect was observed at 250 mg/kg/day.

The Maximum Tolerated Dose (MTD) was found to be below 1000 mg/kg bw/day.

In consultation with the Sponsor, the dose levels considered suitable for a subsequent 28-day toxicity study in the rat were 25, 120 and 600 mg/kg g bw/day.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-10-08 to 2012-11-19 (in-life phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633, from SPF colony
- Age at study initiation: Young adult rats, approximately 7-8 weeks old
- Weight at study initiation: Males: 234-253 g, Females: 174-204 g
- Fasting period before study: no data
- Housing: Standard laboratory conditions during the study, Type III polycarbonate cage, up to 5 animals of the same sex and group/cage.
- Diet (e.g. ad libitum): ssniff® SM R/M-Z+H ad libitum
- Water (e.g. ad libitum): tap water from municipal supply ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.2 – 23.8 °C
- Humidity (%): 30 - 62 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): light 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Route of administration:
oral: gavage
Vehicle:
other: 1% aqueous Methylcellulose
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): 1% aqueous Methylcellulose (abbreviation: 1% MC) was used as vehicle.
- Concentration in vehicle:
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): O16147824 (Dow Chemicals)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test item formulations for concentration and homogeneity was performed two times during the treatment period (during the first and
last weeks of treatment), using a validated ICP method based on the tellurium content.
Duration of treatment / exposure:
28 days
Frequency of treatment:
7 days/week
Remarks:
Doses / Concentrations:
0 mg/kg bw/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
25 mg/kg bw/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
120 mg/kg bw/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
600 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
5 animals/ sex/ dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were set based on available data and information from previous experimental work, including the results of a preliminary dose range finding study
- Rationale for animal assignment (if not random): randomization based on body weights
- Rationale for selecting satellite groups: to assess the reversibility of any changes
- Post-exposure recovery period in satellite groups: 14-day
Positive control:
No positive control
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily. General clinical observations were made daily, after treatment.
- Cage side observations: Animals were inspected for signs of morbidity and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the first treatment and weekly thereafter.
Observation was performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behaviour pattern, changes in gait, posture and response to handling.
Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weight was recorded on Days 0, 7, 14, 18, 21, 25 and 27.
During the recovery period, body weight was measured twice weekly.
Before scheduled necropsy the animals were weighed following overnight food deprivation.

FOOD CONSUMPTION AND COMPOUND INTAKE :
Animal food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g on Day 7 then weekly.
The weekly and mean daily food consumption was calculated.

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 AND COMPOUND INTAKE : No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: collected immediately prior to the scheduled necropsy
- Anaesthetic used for blood collection: Yes (identity): cardiac puncture under pentobarbital anaesthesia
- Animals fasted: Yes, overnight period of food deprivation
- How many animals: all surving animals
- Parameters checked in table below were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: collected immediately prior to the scheduled necropsy
- Animals fasted: Yes, overnight period of food deprivation
- How many animals: all surving animals
- Parameters checked in table below were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During the last week of the treatment period
The examination was repeated on the Recovery Group animals (on Day 39), due to possible changes observed during the Irwin screen conducted on Day 22 and the grip strength measurements in males by the end of the treatment period.
- Dose groups that were examined: each animal
- Battery of functions tested: sensory activity / grip strength / motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table) Gross necropsy was performed on each animal irrespective of the date of death
HISTOPATHOLOGY: Yes (see table) Full histopathology was performed in Control and High Dose Groups animals.
All macroscopic abnormalities in other groups were also examined.
Other examinations:
Examination of vaginal smears
Prior to necropsy, the oestrus cycle of all females was determined by taking vaginal smears, which were prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope, in order to provide information regarding the stage of oestrus cycle at the time
of sacrifice and assist in histological evaluation of oestrogen sensitive tissues.

Statistics:
The statistical analysis was performed using SPSS PC+4.0 software. The heterogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity is found, the normal distribution of data was examined by Kolmogorov-Smirnov test. If the data were not normal distributed, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test.
Recovery groups were compared using Student T-test.
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):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
One High dose female treated at 600 mg/kg bw/day was found dead on Day 24. Clinical signs (hunched posture, piloerection and moderately
decreased activity) and marked body weight loss preceded the death of this animal.

Clinical signs were observed in males and females treated at 120 and 600 mg/kg/day.
At 600 mg/kg/day, clinical signs were noted from Day 3 (males) and Day 2 (females) and included hunched back, piloerection, dark faeces, soft faeces and decreased activity. From Day 10 (4 of 10 males) and Day 22 (1 of 10 female), transient weakness in the hind limbs was also observed. With the exception of dark faeces, the signs were not seen in all animals on each occasion. Following the end of the treatment period the clinical signs ceased although decreased activity, piloerection were still observed for 2-3 days, and hunched posture for 5-7 days. The animals were symptom-free from Day 33 (males) and Day 35 (females).
At 120 mg/kg/day, dark faeces was noted in all animals, transient weakness in the hind limbs was observed only in males (4 of 5) and hunched back was noted occasionally in one male and female.
No clinical signs were observed during the study at 25 mg/kg/day.

BODY WEIGHT AND WEIGHT GAIN
Treatment at 600 mg/kg/day affected body weight gain in both sexes (body weight loss in 3 of 10 males and in 4 of 10 females) resulting in terminal body weights lower by 26-20% and reduction of body weight gain by 66% in males and 90% in females.
Treatment at 120 mg/kg/day caused a reduction in body weight gain by 35% in males and 14% in females. The effect in females was attributed to body weight gain suppression of one animal.
Males at 25 mg/kg/day had slightly lower mean body weight by approximately 8% and overall (Days 0-27) body weight gain by approximately 21%. In one male, slight body weight loss (7%) was noted between Days 25-27. No effect on body weight was observed in females.
Significantly higher body weight gain was observed for both males and females during the 14-day recovery period, indicating reversibility of the effect, however the terminal body weight of males was lower by 15% than control mean.

FOOD CONSUMPTION AND COMPOUND INTAKE
The mean daily food consumption was lower than controls at 120 and 600 mg/kg/day (both sexes) throughout entire treatment period and in males at 25 mg/kg/day during the last week of the treatment period.

HAEMATOLOGY
At 600 mg/kg/day, lower mean platelet count, lower relative lymphocyte count with concomitantly higher relative neutrophil granulocyte count was noted in both males and females and lower relative eosinophil granulocyte count was noted in females.
In females only, a slight decrease in APPT was also noted.
Following the 14-day recovery period slightly increased relative reticulocyte count and higher erythrocyte volume distribution width were recorded in both males and females. In addition, in males slightly lower haematocrit value, slightly lower lymphocyte and higher relative monocyte counts were measured.

CLINICAL CHEMISTRY
Clinical chemistry parameters evaluated on Day 28 were comparable with the controls in all groups of males, except individual values of one male at 600 mg/kg/day. In this animal elevated transaminases (ALT and AST), alkaline phosphatase and gamma GT activity, increased total bilirubin and bile acid concentration and decreased serum creatinine were found.
For females at 600 mg/kg, slightly increased aspartate aminotransferase activity (AST) activity, higher bile acid, slightly higher triglycerides, glucose, urea and sodium concentration was recorded. Slightly higher bile acid and urea concentration was found also at 120 mg/kg.
Following the 14-day recovery period, the above parameters were comparable with the control. Higher phosphorus concentration was measured for both sexes and slightly higher calcium concentration was found in females.

NEUROBEHAVIOUR
During neurological assessment on Day 22, slight differences were recorded for animals treated at 600 mg/kg/day. Findings observed in the Irwin screen included lower performance in the transfer arousal and touch escape response, slightly decreased body tone score or an increased piloerection score in females only and were attributable to poor condition of the animals.
Compared to controls, lower values in grip strength tests were recorded for males and in a few females and were in agreement with observed
transient weakness of the hind limbs. No differences were recorded on Day 39.

ORGAN WEIGHTS
At 600 mg/kg/day higher liver weights (20% in males and 38% in females for body weight relative values) and spleen weights (42-43% for body weight relative values) and lower thymus weights (34-40% for brain relative values) were observed.

GROSS PATHOLOGY
At necropsy on Day 28, black/grey discoloration/foci were observed in a number of organs, including the adrenal gland, brain, stomach, intestines, thymus, mesenteric lymph node and testis. Pale discoloration of the liver in 1 of 5 High dose male, small thymus in 1 of 5 High dose male, 1 of 5 Mid dose female and 2 of 4 High dose females were seen and also corresponded with organ weight changes.

HISTOPATHOLOGY: NON-NEOPLASTIC
Test-item related microscopic findings were observed in the liver, thymus and vagina at a dose level of 600 mg/kg bw.
In the liver, mild or marked (predominantly mild) centrilobular/periportal or diffuse hepatocellular vacuolation was seen in 5 of 5 males and 4 of 4 females associated with mainly minimal mononuclear cell infiltrate. In one male, mild hepatocellular necrosis and increased mitotic activity were also recorded.
In the thymus, mild lymphoid atrophy in 1 of 5 males and 2 of 4 females was in correlation with organ weight changes. Atrophic changes were accompanied with minimal to mild increased apoptosis of lymphocyte in 3 of 5 males and 3 of 4 females. Similar change was observed in one female at 120 mg/kg/day.
The evidence of both findings among animals likely indicated a combined effect of the test item administration (atrophy) and secondary stress (apoptosis) associated with decreased food consumption and body weight gain, increased neutrophils and decreased lymphocytes (stress leukogram) in haematology data of the affected animals.

In 2 of 4 High dose females, moderate diffuse epithelial atrophy of the vagina was noted.


Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
LOAEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male
Basis for effect level:
other: Based on effects on body weight gain at the low dose of 25 mg/kg bw/d no NOAEL could be identified. Lower mean body weight by approximately 8% and overall (Days 0-27) body weight gain by approximately 21%.
Critical effects observed:
not specified

DOSE FORMULATION ANALYSIS

Test item content and homogeneity of the dosing formulations was determined twice during the treatment period (during the first and the last weeks of the treatment). No test item was detected in the Control solution samples. All formulations were shown to be homogeneous. All formulations were found to be in the range of 94 to 104% of nominal concentrations. Based on these results, test item formulations were considered suitable for the study purposes.

Conclusions:
The NOAEL of tellurium dioxide administered by oral gavage to Wistar rats for 28 consecutive days is considered to be the low-dose level of 25 mg/kg/day for females. For males the low dose of 25 mg/kg/day was considered to be the LOAEL, based on reduced body weight gain (21%).

Executive summary:

In a subchronic toxicity study according to OECD guideline 407, tellurium dioxide was administered to groups of 5 Wistar rats by gavage at dose levels of 0, 25, 120 and 600 mg/kg/day. A recovery group was observed for additional 14 days.

Treatment at 600 mg/kg/day caused clinical signs, body weight loss and reduction of mean body weight gain by 66% in males and 90% in females, lower platelet counts in both sexes, black/grey discoloration/foci were observed in the variety of organs, increase in liver and spleen weights and lower thymus weights in both sexes, centrilobular/periportal or diffuse hepatocellular vacuolation associated with mainly minimal mononuclear cell infiltrate in liver, mild lymphoid atrophy accompanied with minimal to mild increased apoptosis of lymphocyte in the thymus, moderate diffuse epithelial atrophy in the vagina. Following 14-day recovery period, signs of reversibility were observed, however no histopathology examination was performed.

Treatment at 120 mg/kg/day was associated with slight clinical signs, including transient weakness of the hind limbs in males, reduction of body weight gain by 35% in males and 14% in females, mild lymphoid atrophy accompanied with minimal to mild increased apoptosis of lymphocyte in thymus in one female.

Treatment at 25 mg/kg/day was associated with lower mean body weight by approximately 8% and overall (Days 0-27) body weight gain by approximately 21% in males.

In conclusion, the NOAEL of tellurium dioxide administered by oral gavage to Wistar rats for 28 consecutive days is considered to be the low-dose level of 25 mg/kg/day for females. For males the low dose of 25 mg/kg/day was considered to be the LOAEL, based on reduced body weight gain (21%).

 

Endpoint:
repeated dose toxicity: oral
Remarks:
other: dose range finder (7days)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
2012-09-18 to 2012-09-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
see read-across justification in section 13.1
Reason / purpose:
read-across source
Qualifier:
no guideline required
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633, from SPF colony
- Age at study initiation: Young adult rats, less than 9 weeks old
- Weight at study initiation:Phase 1: Males: 263-270 g Females: 192-196 g; Phase 2: Males: 231-260 g; Females: 176-215 g
- Fasting period before study: no data
- Housing: Standard laboratory conditions during the study, Type II and III polycarbonate cage, up to 5 animals of the same sex and group/cage.
- Diet (e.g. ad libitum): ssniff® SM R/M-Z+H ad libitum
- Water (e.g. ad libitum): tap water from municipal supply ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.7 – 23.7 °C
- Humidity (%): 31- 63 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): light 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Route of administration:
oral: gavage
Vehicle:
other: 1% aqueous Methylcellulose
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): 1% aqueous Methylcellulose (abbreviation: 1% MC) was used as vehicle.
- Concentration in vehicle:
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of tellurium dioxide dosing formulations for concentration and homogeneity was performed at the Test Site. Top, middle and bottom duplicate samples were taken from test item formulations once during Phase 2, one set to analyze which was collected in replicates and one set as a back-up, if required for any confirmatory analyses. Similarly, one sample was taken in duplicate from the Group 1 (control) solution for concentration measurements. The concentration of tellurium was analysed using ICP/AES.

The achieved concentrations were in the range of 96.7 and 102.8%. No test item was identified in the control samples.
Duration of treatment / exposure:
7 days
Frequency of treatment:
7 days/ week
Remarks:
Doses / Concentrations:
0 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
62.5 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
250 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5 per sex/ dose
Control animals:
yes, concurrent vehicle
Details on study design:
Phase 1 animals were treated by single dose oral gavage administration followed by a 7 day observation period and necropsy. The starting dose level (1000 mg/kg bw) was set by the Sponsor in consultation with the Study Director, based on available data and information from previous experimental work, including the results of an acute oral toxicity study.
Following the administration of 1000 mg/kg dose, all animals survived and no overt sign of toxicity was observed. Based on the results the Phase 2 started at 3 dose levels of 62.5, 250 and 1000 mg/kg bw/day.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Clinical observations were made twice daily, before and after treatment.
Observation was performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behaviour pattern, changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations: on Day 0 and at least on Days 3, 6 and 7 (prior to necropsy, fasted if scheduled euthanasia).

FOOD CONSUMPTION AND COMPOUND INTAKE: yes

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 AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on Day 7, blood samples for clinical pathology evaluation (haematology, coagulation, and clinical biochemistry) were collected immediately prior to the scheduled necropsy, by cardiac puncture under pentobarbital anaesthesia.
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes, After an overnight period of food deprivation of animals
- How many animals: all
- Parameters checked in table below were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on Day 7, blood samples for clinical pathology evaluation (haematology, coagulation, and clinical biochemistry) were collected immediately prior to the scheduled necropsy, by cardiac puncture under pentobarbital anaesthesia.
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table below were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
At the end of Phase 2 on Day 7, blood samples for clinical pathology evaluation (haematology, coagulation, and clinical biochemistry) were collected immediately prior to the scheduled necropsy, by cardiac puncture under pentobarbital anaesthesia.
After an overnight period of food deprivation of animals, 3 blood samples were collected, for haematology (approximately 1.2 mL blood in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), for blood clotting times (approximately 1.4 mL blood for APTT and PT measurements, in tubes with sodium citrate as anticoagulant) and one to obtain serum (approximately 1 mL blood as practical in tubes with no anticoagulant) for clinical chemistry.

On Day 7, all animals were euthanised under pentobarbital anaesthesia by exsanguination and subjected to gross necropsy.
After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.


No histopathology evaluation was performed.
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):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study. No clinical signs were observed during the study. All animals were symptom-free.
Dark faeces were observed in all animals at 250 and 1000 mg/kg/day on Day 6.

BODY WEIGHT AND WEIGHT GAIN
Body weight gain suppression was observed in both sexes at 1000 mg/kg/day, resulting in terminal body weight lower by approximately 4% (males) and 8% (females), compared to the control mean. In 4 of 5 females, negligible body weight gain or slight body weight loss were observed.
There were no toxicologically significant or adverse effects observed on the body weight following the administration of the test item at doses of 62.5 and 250 mg/kg/day for 7 consecutive days.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Lower food consumption was recorded for both males and females at 1000 mg/kg/day during the entire treatment period. Compared to the control mean, the mean values were lower by approximately 8% in males and 28% in females.
There were no adverse effects on the animal food consumption or differences ascribed to test item administration at doses of 62.5 and 250 mg/kg/day.

HAEMATOLOGY
Compared to controls, mean relative reticulocyte count was lower in both males and females at 1000 mg/kg/day. The differences were statistically significant (p<0.01). All values were within the normal range.
In females at 1000 mg/kg/day mean platelet count was significantly lower, when compared to the control mean. Lower platelet count was found in 4 of 5 females. (Table 2). Slightly lower mean platelet volume (MPV) was also measured for High dose females, however, individual values were not proportional to the individual platelet count.
Compared to control means, slightly higher relative monocyte count was measured in females treated at 62.5 mg/kg/day. The difference attained statistical significance (p<0.01).

CLINICAL CHEMISTRY
No test item related adverse effects at any dose level were noted in clinical chemistry parameters. All parameters were within the normal physiological ranges for the age, strain and sex of rats on test.

ORGAN WEIGHTS
The mean absolute and relative thymus weights adjusted for both brain and terminal body weight were significantly lower than the controls in females treated at 1000 mg/kg/day. The decrease was in the range of 33% for absolute value and 29-35% for relative values and were statistically significant (p<0.05 for absolute and body weigh relative values, p<0.01 for brain relative values)
Higher than control liver weights were noted at 1000 mg/kg/day in females. The increase was approximately of 13% for body weight relative weights and attained statistical significance (p<0.05).
Compared to the control mean, the absolute and relative weights of kidneys were lower in males at 1000 mg/kg/day by approximately 16% (absolute values) and the difference were statistically significant (p<0.05 for absolute and, p<0.01 for relative values). Lower relative weights of kidneys were also noted in the Low and Mid dosed males (p<0.05).

GROSS PATHOLOGY
There was no evidence of test item-related macroscopic findings in animals at necropsy following a 7-day administration of the test item, with the exception of black discoloration of the digestive content of stomach and intestines noted in males at 250 and 1000 mg/kg/day and in all treated groups of females.

Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: 7 days exposure
Critical effects observed:
not specified
Conclusions:
The Maximum Tolerated Dose (MTD) was found to be below 1000 mg/kg bw/day.
No adverse effect was observed at 250 mg/kg/day.
Executive summary:

Administration of tellurium dioxide to Wistar rats by oral gavage, daily for 7 days, at a dose level of 1000 mg/kg bw/day was associated with body weight gain suppression in both sexes, mild effect in clinical chemistry parameters (lower relative reticulocyte counts in both sexes, lower platelet counts and higher serum calcium concentration in females), an lower kidney weights in males and lower thymus weight in females. No adverse effect was observed at 250 mg/kg/day.

The Maximum Tolerated Dose (MTD) was found to be below 1000 mg/kg bw/day.

In consultation with the Sponsor, the dose levels considered suitable for a subsequent 28-day toxicity study in the rat were 25, 120 and 600 mg/kg g bw/day.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2012-10-08 to 2012-11-19 (in-life phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
see justification for read-across in section 13.1
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633, from SPF colony
- Age at study initiation: Young adult rats, approximately 7-8 weeks old
- Weight at study initiation: Males: 234-253 g, Females: 174-204 g
- Fasting period before study: no data
- Housing: Standard laboratory conditions during the study, Type III polycarbonate cage, up to 5 animals of the same sex and group/cage.
- Diet (e.g. ad libitum): ssniff® SM R/M-Z+H ad libitum
- Water (e.g. ad libitum): tap water from municipal supply ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.2 – 23.8 °C
- Humidity (%): 30 - 62 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): light 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Route of administration:
oral: gavage
Vehicle:
other: 1% aqueous Methylcellulose
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): 1% aqueous Methylcellulose (abbreviation: 1% MC) was used as vehicle.
- Concentration in vehicle:
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): O16147824 (Dow Chemicals)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test item formulations for concentration and homogeneity was performed two times during the treatment period (during the first and
last weeks of treatment), using a validated ICP method based on the tellurium content.
Duration of treatment / exposure:
28 days
Frequency of treatment:
7 days/week
Remarks:
Doses / Concentrations:
0 mg/kg bw/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
25 mg/kg bw/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
120 mg/kg bw/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
600 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
5 animals/ sex/ dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were set based on available data and information from previous experimental work, including the results of a preliminary dose range finding study
- Rationale for animal assignment (if not random): randomization based on body weights
- Rationale for selecting satellite groups: to assess the reversibility of any changes
- Post-exposure recovery period in satellite groups: 14-day
Positive control:
No positive control
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily. General clinical observations were made daily, after treatment.
- Cage side observations: Animals were inspected for signs of morbidity and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the first treatment and weekly thereafter.
Observation was performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behaviour pattern, changes in gait, posture and response to handling.
Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weight was recorded on Days 0, 7, 14, 18, 21, 25 and 27.
During the recovery period, body weight was measured twice weekly.
Before scheduled necropsy the animals were weighed following overnight food deprivation.

FOOD CONSUMPTION AND COMPOUND INTAKE :
Animal food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g on Day 7 then weekly.
The weekly and mean daily food consumption was calculated.

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 AND COMPOUND INTAKE : No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: collected immediately prior to the scheduled necropsy
- Anaesthetic used for blood collection: Yes (identity): cardiac puncture under pentobarbital anaesthesia
- Animals fasted: Yes, overnight period of food deprivation
- How many animals: all surving animals
- Parameters checked in table below were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: collected immediately prior to the scheduled necropsy
- Animals fasted: Yes, overnight period of food deprivation
- How many animals: all surving animals
- Parameters checked in table below were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During the last week of the treatment period
The examination was repeated on the Recovery Group animals (on Day 39), due to possible changes observed during the Irwin screen conducted on Day 22 and the grip strength measurements in males by the end of the treatment period.
- Dose groups that were examined: each animal
- Battery of functions tested: sensory activity / grip strength / motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table) Gross necropsy was performed on each animal irrespective of the date of death
HISTOPATHOLOGY: Yes (see table) Full histopathology was performed in Control and High Dose Groups animals.
All macroscopic abnormalities in other groups were also examined.
Other examinations:
Examination of vaginal smears
Prior to necropsy, the oestrus cycle of all females was determined by taking vaginal smears, which were prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope, in order to provide information regarding the stage of oestrus cycle at the time
of sacrifice and assist in histological evaluation of oestrogen sensitive tissues.

Statistics:
The statistical analysis was performed using SPSS PC+4.0 software. The heterogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity is found, the normal distribution of data was examined by Kolmogorov-Smirnov test. If the data were not normal distributed, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test.
Recovery groups were compared using Student T-test.
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):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
One High dose female treated at 600 mg/kg bw/day was found dead on Day 24. Clinical signs (hunched posture, piloerection and moderately
decreased activity) and marked body weight loss preceded the death of this animal.

Clinical signs were observed in males and females treated at 120 and 600 mg/kg/day.
At 600 mg/kg/day, clinical signs were noted from Day 3 (males) and Day 2 (females) and included hunched back, piloerection, dark faeces, soft faeces and decreased activity. From Day 10 (4 of 10 males) and Day 22 (1 of 10 female), transient weakness in the hind limbs was also observed. With the exception of dark faeces, the signs were not seen in all animals on each occasion. Following the end of the treatment period the clinical signs ceased although decreased activity, piloerection were still observed for 2-3 days, and hunched posture for 5-7 days. The animals were symptom-free from Day 33 (males) and Day 35 (females).
At 120 mg/kg/day, dark faeces was noted in all animals, transient weakness in the hind limbs was observed only in males (4 of 5) and hunched back was noted occasionally in one male and female.
No clinical signs were observed during the study at 25 mg/kg/day.

BODY WEIGHT AND WEIGHT GAIN
Treatment at 600 mg/kg/day affected body weight gain in both sexes (body weight loss in 3 of 10 males and in 4 of 10 females) resulting in terminal body weights lower by 26-20% and reduction of body weight gain by 66% in males and 90% in females.
Treatment at 120 mg/kg/day caused a reduction in body weight gain by 35% in males and 14% in females. The effect in females was attributed to body weight gain suppression of one animal.
Males at 25 mg/kg/day had slightly lower mean body weight by approximately 8% and overall (Days 0-27) body weight gain by approximately 21%. In one male, slight body weight loss (7%) was noted between Days 25-27. No effect on body weight was observed in females.
Significantly higher body weight gain was observed for both males and females during the 14-day recovery period, indicating reversibility of the effect, however the terminal body weight of males was lower by 15% than control mean.

FOOD CONSUMPTION AND COMPOUND INTAKE
The mean daily food consumption was lower than controls at 120 and 600 mg/kg/day (both sexes) throughout entire treatment period and in males at 25 mg/kg/day during the last week of the treatment period.

HAEMATOLOGY
At 600 mg/kg/day, lower mean platelet count, lower relative lymphocyte count with concomitantly higher relative neutrophil granulocyte count was noted in both males and females and lower relative eosinophil granulocyte count was noted in females.
In females only, a slight decrease in APPT was also noted.
Following the 14-day recovery period slightly increased relative reticulocyte count and higher erythrocyte volume distribution width were recorded in both males and females. In addition, in males slightly lower haematocrit value, slightly lower lymphocyte and higher relative monocyte counts were measured.

CLINICAL CHEMISTRY
Clinical chemistry parameters evaluated on Day 28 were comparable with the controls in all groups of males, except individual values of one male at 600 mg/kg/day. In this animal elevated transaminases (ALT and AST), alkaline phosphatase and gamma GT activity, increased total bilirubin and bile acid concentration and decreased serum creatinine were found.
For females at 600 mg/kg, slightly increased aspartate aminotransferase activity (AST) activity, higher bile acid, slightly higher triglycerides, glucose, urea and sodium concentration was recorded. Slightly higher bile acid and urea concentration was found also at 120 mg/kg.
Following the 14-day recovery period, the above parameters were comparable with the control. Higher phosphorus concentration was measured for both sexes and slightly higher calcium concentration was found in females.

NEUROBEHAVIOUR
During neurological assessment on Day 22, slight differences were recorded for animals treated at 600 mg/kg/day. Findings observed in the Irwin screen included lower performance in the transfer arousal and touch escape response, slightly decreased body tone score or an increased piloerection score in females only and were attributable to poor condition of the animals.
Compared to controls, lower values in grip strength tests were recorded for males and in a few females and were in agreement with observed
transient weakness of the hind limbs. No differences were recorded on Day 39.

ORGAN WEIGHTS
At 600 mg/kg/day higher liver weights (20% in males and 38% in females for body weight relative values) and spleen weights (42-43% for body weight relative values) and lower thymus weights (34-40% for brain relative values) were observed.

GROSS PATHOLOGY
At necropsy on Day 28, black/grey discoloration/foci were observed in a number of organs, including the adrenal gland, brain, stomach, intestines, thymus, mesenteric lymph node and testis. Pale discoloration of the liver in 1 of 5 High dose male, small thymus in 1 of 5 High dose male, 1 of 5 Mid dose female and 2 of 4 High dose females were seen and also corresponded with organ weight changes.

HISTOPATHOLOGY: NON-NEOPLASTIC
Test-item related microscopic findings were observed in the liver, thymus and vagina at a dose level of 600 mg/kg bw.
In the liver, mild or marked (predominantly mild) centrilobular/periportal or diffuse hepatocellular vacuolation was seen in 5 of 5 males and 4 of 4 females associated with mainly minimal mononuclear cell infiltrate. In one male, mild hepatocellular necrosis and increased mitotic activity were also recorded.
In the thymus, mild lymphoid atrophy in 1 of 5 males and 2 of 4 females was in correlation with organ weight changes. Atrophic changes were accompanied with minimal to mild increased apoptosis of lymphocyte in 3 of 5 males and 3 of 4 females. Similar change was observed in one female at 120 mg/kg/day.
The evidence of both findings among animals likely indicated a combined effect of the test item administration (atrophy) and secondary stress (apoptosis) associated with decreased food consumption and body weight gain, increased neutrophils and decreased lymphocytes (stress leukogram) in haematology data of the affected animals.

In 2 of 4 High dose females, moderate diffuse epithelial atrophy of the vagina was noted.


Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
LOAEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male
Basis for effect level:
other: Based on effects on body weight gain at the low dose of 25 mg/kg bw/d no NOAEL could be identified. Lower mean body weight by approximately 8% and overall (Days 0-27) body weight gain by approximately 21%.
Critical effects observed:
not specified

DOSE FORMULATION ANALYSIS

Test item content and homogeneity of the dosing formulations was determined twice during the treatment period (during the first and the last weeks of the treatment). No test item was detected in the Control solution samples. All formulations were shown to be homogeneous. All formulations were found to be in the range of 94 to 104% of nominal concentrations. Based on these results, test item formulations were considered suitable for the study purposes.

Conclusions:
The NOAEL of tellurium dioxide administered by oral gavage to Wistar rats for 28 consecutive days is considered to be the low-dose level of 25 mg/kg/day for females. For males the low dose of 25 mg/kg/day was considered to be the LOAEL, based on reduced body weight gain (21%).

Executive summary:

In a subchronic toxicity study according to OECD guideline 407, tellurium dioxide was administered to groups of 5 Wistar rats by gavage at dose levels of 0, 25, 120 and 600 mg/kg/day. A recovery group was observed for additional 14 days.

Treatment at 600 mg/kg/day caused clinical signs, body weight loss and reduction of mean body weight gain by 66% in males and 90% in females, lower platelet counts in both sexes, black/grey discoloration/foci were observed in the variety of organs, increase in liver and spleen weights and lower thymus weights in both sexes, centrilobular/periportal or diffuse hepatocellular vacuolation associated with mainly minimal mononuclear cell infiltrate in liver, mild lymphoid atrophy accompanied with minimal to mild increased apoptosis of lymphocyte in the thymus, moderate diffuse epithelial atrophy in the vagina. Following 14-day recovery period, signs of reversibility were observed, however no histopathology examination was performed.

Treatment at 120 mg/kg/day was associated with slight clinical signs, including transient weakness of the hind limbs in males, reduction of body weight gain by 35% in males and 14% in females, mild lymphoid atrophy accompanied with minimal to mild increased apoptosis of lymphocyte in thymus in one female.

Treatment at 25 mg/kg/day was associated with lower mean body weight by approximately 8% and overall (Days 0-27) body weight gain by approximately 21% in males.

In conclusion, the NOAEL of tellurium dioxide administered by oral gavage to Wistar rats for 28 consecutive days is considered to be the low-dose level of 25 mg/kg/day for females. For males the low dose of 25 mg/kg/day was considered to be the LOAEL, based on reduced body weight gain (21%).

 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat

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

In a toxicity study according to OECD guideline 407, tellurium dioxide was administered to groups of 5 Wistar rats by gavage at dose levels of 0, 25, 120 and 600 mg/kg/day.A recovery group was observed for additional 14 days.

Treatment at 600 mg/kg/day caused clinical signs, body weight loss and reduction of mean body weight gain by 66% in males and 90% in females, lower platelet counts in both sexes, black/grey discoloration/foci were observed in the variety of organs, increase in liver and spleen weights and lower thymus weights in both sexes, centrilobular/periportal or diffuse hepatocellular vacuolation associated with mainly minimal mononuclear cell infiltrate in liver, mild lymphoid atrophy accompanied with minimal to mild increased apoptosis of lymphocyte in the thymus, moderate diffuse epithelial atrophy in the vagina. Following 14-day recovery period, signs of reversibility were observed, however no histopathology examination was performed.

Treatment at 120 mg/kg/day was associated with slight clinical signs, including transient weakness of the hind limbs in males, reduction of body weight gain by 35% in males and 14% in females, mild lymphoid atrophy accompanied with minimal to mild increased apoptosis of lymphocyte in thymus in one female.

Treatment at 25 mg/kg/day was associated with lower mean body weight by approximately 8% and overall (Days 0-27) body weight gain by approximately 21% in males.

In conclusion, the NOAEL of tellurium dioxide administered by oral gavage to Wistar rats for 28 consecutive days is considered to be the low-dose level of 25 mg/kg/day for females. For males the low dose of 25 mg/kg/day was considered to be the LOAEL, based on reduced body weight gain (21%).

In the subsequent 90-day oral toxicity study (OECD 408), Tellurium dioxide was administered to groups of 10 Wistar rats (males and females) by gavage at dose levels of 0, 10, 30 and 100 mg/kg/day. At the highest dose and the control, an additional recovery group with 5 animals was observed for 4 weeks.

Slight signs of treatment-related effets (clinical signs, reduction in body weight and food consumption) were observed during the in-vivo phase of the study, mainly at the high dose level of 100mg/kg/day and, with a lower extent at the mid-dose level of 30 mg/kg/day. At microscopic examination, changes were observed at the end of the treatment period in the liver of animals dosed at ≥ 10 mg/kg/day. However, these changes were no longer present at the end of the 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 was concluded that the NOAEL for this study is 100 mg/kg/day.

For extrapolation of a NAEL for repeated dose toxicity refer to the attached Expert Statement.

Justification for read-across is outlined in the attached document.

Justification for classification or non-classification