Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 April 2012 - 09 October 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1,3-diphenyl-2-thiourea
EC Number:
203-004-2
EC Name:
1,3-diphenyl-2-thiourea
Cas Number:
102-08-9
Molecular formula:
C13H12N2S
IUPAC Name:
1,3-diphenylthiourea
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: breeder: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approximately 6 weeks old on the first day of treatment
- Mean body weight at study initiation: the males had a mean body weight of 249 g (range: 237 g to 263 g) and the females had a mean body weight of 202 g (range: 185 g to 214 g)
- Fasting period before study: no
- Housing: the animals were housed by five from the same sex and group in polycarbonate cages with stainless steel lids
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: at least 9 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 20 April 2012 to 18 May 2012.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a homogeneous suspension in the vehicle.
The test item was ground to a fine powder, using a mortar and pestle, and then mixed with the required quantity of vehicle.
The frequency of dosage formulation preparation was based on available stability data.
The dosage forms were stored at room temperature and protected from light and delivered to the study room in brown flasks.

VEHICLE
- Concentration in vehicle: 10, 50 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: HPLC-UV.
Test item concentrations: remained within an acceptable range of variation compared to nominal values.
Homogeneity: homogenous
Stability: stable after 9 days at room temperature and protected from light
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 animals per sex per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor based on the results of a 14-day toxicity study. In this preliminary study, the test item was given at 50, 250 or 1000 mg/kg/day to Sprague-Dawley rats. No unscheduled mortalities occurred during the treatment period. Ptyalism was observed sporadically in test item-treated animals. Slight soft feces was noted in 1/3 males at 1000 mg/kg/day from day 8 to day 11. There was a reduction in mean food consumption in males at 1000 mg/kg/day, related with lower mean body weight gain, principally during the first week of treatment. There were no test item related macroscopic findings.

- Rationale for animal assignment: computerized stratification procedure.
Positive control:
no (not required)

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
- Time schedule: for mortality,once a day during the acclimation period and at least twice a day during the treatment period. For clinical signs, once a day.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: at the beginning of the treatment period and then once a week until the end of the study.

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

FOOD CONSUMPTION:
- Time schedule: once a week until the end of the study.

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule: each animal was evaluated once in week 4.

HAEMATOLOGY, CLINICAL CHEMISTRY, URINALYSIS:
- Time schedule: at the end of the treatment period.
Sacrifice and pathology:
ORGAN WEIGHTS: see table below


GROSS PATHOLOGY:
Complete macroscopic post-mortem examination of all study animals


HISTOPATHOLOGY:
- on all tissues listed in the table below for the control and high dose animals (groups 1 and 4) sacrificed at the end of the treatment period,
- on all macroscopic lesions from all low- and intermediate-dose animals (groups 2 and 3) sacrificed on completion of the treatment period.

Based upon the microscopic results of the high-dose group and in agreement of the Sponsor, the following tissues from the low- and intermediate-dose animals were examined: liver, thyroids, thymus, spleen, heart, lungs, adrenals, kidneys, stomach and bone marrow.
Other examinations:
no
Statistics:
Citox software (version D.6) (see § Study plan adherence) was used to perform the statistical analyses of body weight, hematology, blood biochemistry, thyroid hormones and urinalysis data. PathData software (version 6.2d2) was used to perform the statistical analysis of organ weight data (level of significance: 0.05 or 0.01)

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 50 and 250 mg/kg/day, no clinical signs related to the test item treatment were recorded.
At 1000 mg/kg/day, thin appearance was observed from day 6 to day 11 in 3/5 females. One of these females had with a body weight loss of 12% between days 1 and 8 and presented hunched posture from day 22. These findings were considered to be related to treatment with the test item.

Ptyalism was observed mostly from day 6 in test item-treated animals without dose-relationship. This finding is commonly observed when a test item is administered by gavage and was not considered as an adverse effect.
Incidental findings included scabs, wound, thinning of hair and alopecia were observed in control and test item-treated isolated animals. These clinical signs were noted with low incidence and without dose-relationship and can occur spontaneously in untreated laboratory rats of this strain and age. They were considered to be unrelated to the test item treatment.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 50 and 250 mg/kg/day, body weight was not affected by the test item treatment.
At 1000 mg/kg/day, when compared to mean control values, lower mean body weight and mean body weight gain were observed in males and females from week 1, resulting in a final body weight statistically significantly lower than mean control values.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food intake was not affected by the test item treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Lower neutrophils (-40%; p<0.05), eosinophils (-78%; p<0.01) and monocytes ( 51%; p<0.01) counts were observed in high-dose males and lower mean white blood cell count (-42%; p<0.05), due to lower eosinophils (-76%; p<0.05) and basophils (-67%; p<0.05) counts, were noted in high dose females when compared to control values. These changes in hematology parameters were considered to be related to treatment with the test item.

Lower mean red blood cell count (-10% and -17%), associated with lower mean hemoglobin concentration (-9% and -22%) and correlated with lower mean packed cell volume (-11% and 20%), were observed in high dose males and females (mostly in females) when compared to control values. A trend to a decrease mean red blood cell count, hemoglobin concentration and packed cell volume was observed in mid-dose animals. These minor to moderate changes were considered to be related to the treatment with the test item.

Other findings (namely: shortened prothrombin time in high-dose animals and higher thrombocyte concentration in mid-dose males) were either without biologically significance, or without a dose relationship, or without correlation with other red blood cell parameters, and they were therefore considered not to be test item-related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Higher mean total protein concentration (+7% in males and +19% in females), correlating with higher mean albumin concentrations (+9% in males and +21% in females) and mean calcium concentration (+4% in males and +10% in females), was noted in high-dose animals (mostly in females), when compared to control values. In addition, dose-related higher mean cholesterol level was noted from 50 mg/kg/day in both sexes when compared to control values (1.35-fold p<0.05 to 3.15-fold; p<0.01). These findings could be correlated with the changes in the thyroids hormones metabolism.
The other findings (namely: higher creatinine, higher potassium, lower chloride and lower alkaline phosphatase activity) were considered not to be test item treatment related as they were either not dose-related or not biologically significant, or they were of minimal amplitude or not consistent in males and females.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinary parameters were not disturbed by the administration of the test item.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no test item-related effects at Functional Observation Battery, including motor activity.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Increased liver and thyroid weights correlated with enlargement at macroscopic examination (thyroids) and with hypertrophy (liver, thyroid) at microscopic examination.
Decreased thymus, adrenal and spleen weights correlated with reduced size at macroscopic examination (thymus, adrenals) and with lymphoid atrophy (thymus, spleen) and cortical atrophy (adrenals) at microscopic examination. These weight decreases were considered to be due to the body weight decrease in treated animals.
Decreased prostate and seminal vesicle weights were without correlating microscopic findings, and were therefore considered unlikely to be treatment-related.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Enlarged thyroids correlated with hypertrophy at microscopic examination.
Reduced size of the thymus and adrenals correlated with histopathological findings of atrophy.
There were no other treatment-related macroscopic findings. Incidental findings were typical of this strain and age of rat.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings considered to be due to direct effects of the test compound were noted in the liver, thyroids, and brown fat:
-Liver: hepatocyte hypertrophy was characterised by diffuse enlargement of hepatocytes, with a minimal increase in basophilic stippling of cytoplasm.
-Thyroids: The changes in the liver and thyroids were consistent with liver enzyme induction and secondary thyroid hypertrophy.
-Brown fat: Treatment-related findings considered to be secondary to cachexia, rather than a direct effect of the test compound were noted in the thymus, spleen, adrenals, stomach, and bone marrow:
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Thyroid hormones
No variation in T3 level was observed in test item-treated males when compared to control values.
Higher mean T3 level was noted in mid- and high-dose females (+27% and +23%, respectively), when compared to control values. Nevertheless, this variation was not observed in males and remained within the range of values commonly observed in untreated animals of the same age and strain. It was therefore considered to be of no toxicological significance.
Dose-related decreased mean T4 level (-24 to -56%) and increased mean TSH level (+3-fold to +15-fold) were noted from 50 mg/kg/day in both sexes when compared to control values, reaching statistically significance at 1000 mg/kg/day for T4 level and from 250 mg/kg/day for TSH level. These findings correlated with hypertrophy at microscopic examination.

Effect levels

Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: adverse effects observed on thyroid (enlarged thyroid, thyroid follicular hypertrophy, statistically significant increased TSH level) and liver (increase in weight, hepatocyte hypertrophy) observed at 250 mg/kg/day in male and female rats.

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (actual dose received)
System:
other: thyroid gland
Organ:
thyroid gland
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
The test item was administered daily for 4 weeks by the oral route to male and female Sprague-Dawley rats at dose-levels of 50, 250 or 1000 mg/kg/day in corn oil. Treatment with the test item resulted in some systemic toxicity (some clinical signs, reducing in body weight, low white and red blood cells count and high total protein, albumin and calcium concentrations) at 1000 mg/kg/day. A trend to a decrease red blood cell parameters were observed at 250 mg/kg/day. Dose-related higher cholesterol level was noted from 50 mg/kg/day. Dose related decreased T4 level and increased TSH level were noted from 250 mg/kg/day.
Macroscopic and microscopic changes in the liver from 250 mg/kg/day and thyroids from 50 mg/kg/day were considered to be due to direct effects of the test compound, and were consistent with liver enzyme induction and secondary to the thyroid hypertrophy.

The No Observed Adverse Effect Level (NOAEL) was considered to be at 50 mg/kg/day based on the adverse effects observed on thyroid (enlarged thyroid, thyroid follicular hypertrophy, statistically significant increased TSH level) and liver (increase in weight, hepatocyte hypertrophy) observed at 250 mg/kg/day in male and female rats.
Executive summary:

The objective of this study was to evaluate the potential toxicity of the test item following daily oral administration (gavage) to rats for4 weeks.

This study was performed following the OECD guideline No. 407 and the principles of Good Laboratory Practices.

 

Methods

Three groups of five male and five female Sprague-Dawley rats received the test item by daily oral administration for 28 days at dose-levels of 50, 250 or 1000 mg/kg/day. The test item was administered as a homogenous suspension in the vehicle (corn oil) at a constant dosage-volume of 5 mL/kg/day. A control group of five males and five females received the vehicle alone under the same experimental conditions.

Test item concentrations were checked on formulations used in weeks 1 and 4.

The health status of the animals was checked and detailed clinical examinations were performed at least once weekly.In addition, a Functional Observation Battery was evaluated in each animal once in week 4.

Body weight was recorded once before the beginning of the treatment period, on the first day of treatment and then at least once a week during the study.The quantity of food consumed by the animals in each cage was recorded at the same time as body weight.

Hematology, blood biochemistry and urinalysis were performed on all animalsat the end of the treatment period. The levels of the thyroid hormones (T3 and T4) and thyroid stimulating hormone (TSH) were determined on all animals at the end of the treatment period.

On completion of the treatment period, the animals were euthanized and submitted to a full macroscopic post-mortem examination. Designated organs were weighed and selected tissues were preserved. A microscopic examination was performed on selected tissues from the control- and high-dose animals sacrificed at the end of the treatment period and on all macroscopic lesions from all low- and intermediate-dose animals.Based upon the microscopic results of the high-dose group, the liver, thyroids, thymus, spleen, heart, lungs, adrenals, kidneys, stomach and bone marrowwere examined from the low- and intermediate-dose animals.

 

Results

The test item concentrations in the administered dose formulations analyzed in weeks 1 and 4 were within the acceptance criteria.

 

There were no unscheduled deaths that were related to test item treatment.

 

At 1000 mg/kg/day, thin appearance was observed from day 6 to day 11 in 3/5 females, along with a body weight loss of 12% between days 1 and 8 and hunched posture from day 22 one of these females. No relevant clinical signs were recorded in animals treated at 50 or 250 mg/kg/day.

 

Lower body weight and body weight gain were observed in high-dose animals from week 1, resulting in a lower body weight at the end of the treatment period.Body weight of animals given 50 or 250 mg/kg/day was not affected by the test item treatment.

 

Food intake was not affected by the test item treatment. There were no test item-related effects on FOB assays, including motor activity.


Lower neutrophils, eosinophils and monocytes counts were observed in high-dose males and lower mean white blood cell count, due to lower eosinophils and basophils counts, were noted in high-dose females when compared to control values.

In high-dose animals, decreased in red blood cell parameters were observed (mostly in females). A trend to a decrease mean red blood cell parameters were observed in mid-dose animals.

 

Higher mean total protein concentration, correlating with higher mean albumin concentration and higher mean calcium concentration, was noted in high-dose animals (more marked in females than in males) when compared to control values.In addition, dose-related higher mean cholesterol level was noted from 50 mg/kg/day in both sexes when compared to control values. These findings could be correlated withthe changes in the thyroids hormones metabolism.

 

Urinary parameters were not disturbed by the administration of the test item.

 

Dose-related increased mean T3 level was observed from 250 mg/kg/day in females but not in males. Dose-related decreased mean T4 level and increased mean TSH level were noted from 250 mg/kg/day in both sexes when compared to control values, reaching statistically significance at 1000 mg/kg/day for T4 level and from 250 mg/kg/day for TSH level. These findings correlated with hypertrophy at microscopic examination.

 

The following treatment-related body/organ weight changes were present:

. decrease in body weight in males and females receiving 1000 mg/kg/day,

. increase in weight of liver (males and females at 250 mg/kg/day and above) and thyroid (males and females at 1000 mg/kg/day),

. decrease in weight of thymus (males and females at 50 mg/kg/day and above), spleen (males and females at 1000 mg/kg/day) and adrenals (females at 1000 mg/kg/day).

 

The following treatment-related necropsy findings were present:

. enlarged thyroids in males and females at 250 mg/kg/day and above,

. reduced size of thymus (males at 1000 mg/kg/day, females at 250 mg/kg/day and above),

. reduced size of adrenals (males and females at 1000 mg/kg/day).

 

The following treatment-related microscopic findings were present:

. liver hepatocyte hypertrophy (males and females at 250 mg/kg/day and above),

. thyroid follicular hypertrophy (males at 250 mg/kg and above, females at 50  mg/kg/day and above),

. degeneration and/or inflammation of brown fat in various organs (males and females at 50 mg/kg/day and above),

. lymphoid atrophy in thymus (males at 50 mg/kg/day and above, females at 250 mg/kg/day and above) and in spleen (males and females at 1000 mg/kg/day),

. adrenal cortical atrophy (males and females at 1000 mg/kg/day),

. stomach erosion (males and females at 1000 mg/kg/day),

. reduced cellularity of bone marrow (females at 1000 mg/kg/day).

 

The changes in the liver, thyroids and brown fat were considered to be due to direct effects of the test compound. Liver and thyroid effects wereconsistent with liver enzyme induction and secondary thyroid hypertrophy. Atrophy in thethymus, spleen, and adrenals, erosion in the stomach, and reduced cellularity of bone marrow were considered to be secondary to cachexia.

 

Based on increased mean TSH level, macroscopic and microscopic findings on liver and thyroid (males and females) at 250 mg/kg/day, the NOAEL (No Observable Adverse Effect Level) was considered to be at 50 mg/kg/day.


Based on higher mean cholesterol level, decreases weight of thymus (males and females), degeneration and/or inflammation of brown fat (males and females), lymphoid atrophy (males), and thyroid follicular hypertrophy (females) at 50 mg/kg/day, the lowest dosage evaluated in this study, a NOEL (No Observable Effect Level) could not be determined.

 

Conclusion

The test item was administered daily for 4 weeks by the oral route to male and female Sprague-Dawley rats at dose-levels of 50, 250 or 1000 mg/kg/dayincorn oil.

 

Treatment with the test item resulted in some systemic toxicity (some clinical signs, reducing in body weight, low white and red blood cellscountand high total protein, albumin and calcium concentrations) at 1000 mg/kg/day. A trend to a decrease red blood cell parameters were observed at 250 mg/kg/day. Dose-related higher cholesterol level was noted from 50 mg/kg/day. Dose-related decreased T4 level and increased TSH level were noted from 250 mg/kg/day.

Macroscopic and microscopic changes in the liver from 250 mg/kg/day and thyroids from 50 mg/kg/day were considered to be due to direct effects of the test compound, and wereconsistent with liver enzyme induction and secondary to the thyroid hypertrophy.

 

The No Observed Adverse Effect Level (NOAEL) was considered to be at 50 mg/kg/day based on the adverse effects observed on thyroid (enlarged thyroid, thyroid follicular hypertrophy, statistically significant increased TSH level) and liver (increase in weight, hepatocyte hypertrophy) observed at 250 mg/kg/day in male and female rats.