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Administrative data

Description of key information

Only one repeated study in available : the test item was administered daily for 4 weeks by the oral route to male and female Sprague-Dawley rats at dose-levels of 0.5, 2.5 or 10 mg/kg/day. The No Observed Adverse Effect Level (NOAEL) was considered to be 2.5 mg/kg/day based on effects observed at 10 mg/kg/day on thymus, spleen and bone marrow.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 November 2011 - 24 February 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
USA EPA Health Effects Test Guideline OPPTS 870.3050, July 2000
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder:Charles River Laboratories France, l’Arbresle, 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 193 g (range: 170 g to 213 g) and the females had a mean body weight of 148 g (range: 133 g to 165 g)
- Fasting period before study: no
- Housing: the animals were housed by five, 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 7 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 (7:00 - 19:00)

IN-LIFE DATES: 17 November 2011 to 15 December 2011.
Route of administration:
oral: gavage
Vehicle:
other: drinking water treated by reverse osmosis
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a solution in the vehicle. The test item was mixed with the required quantity of vehicle.

The frequency of dose formulation preparation was based on available stability data. The dose formulations were stored at room temperature and protected from light pending delivery to the study room in brown flasks.

VEHICLE
- Concentration in vehicle: 0.1, 0.5 and 2 mg/mL
- Amount of vehicle: 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:
The test item concentrations in the administered dose formulations analyzed in weeks 1 and 4 remained within an acceptable range of +0.3% to +3.3% when compared to the nominal values (± 10%).
Homogeneity: not assessed, dose formulation is a solution
Stability: performed in another study (0.1 and 3 mg/mL stable after 9 days storage at room temperature, protected from light in vehicle).
Duration of treatment / exposure:
4 weeks.
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0, 0.5, 2.5 and 10 mg/kg/day
Basis:
actual ingested
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 an oral acute LD50 of 15 mg/kg and the results of a preliminary 14-day study in the same conditions (administration by gavage in aqueous solution). No effects were observed (clinical signs, body weight and macroscopic observation) at 0.5, 2 and 8 mg/kg/day in rats.

- Rationale for animal assignment: computerized stratification procedure.
Positive control:
no (not required)
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
- Time schedule: once a day during the acclimation period and at least twice a day (mortality) or once a day (clinical signs) during the treatment period.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: once before 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 at the end of the treatment period.

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

GROSS PATHOLOGY:
A complete macroscopic post-mortem examination was performed on all study animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

HISTOPATHOLOGY:
A microscopic examination was performed on:
- all tissues listed in the Tissue Procedure Table from the control and high-dose animals (groups 1 and 4) sacrificed at the end of the treatment period,
- spleen, bone marrow and thymus of the low- and intermediate-dose animals (groups 2 and 3) sacrificed at the end of the treatment period,
- all macroscopic lesions from all low- and intermediate-dose animals (groups 2 and 3) sacrificed on completion of the treatment period.
Other examinations:
no
Statistics:
Citox solfware was used to perform the statistical analysses of body weight, hematology, blood chemistry and urinalysis.
PathData sotware was used to perform the statistical analysis of organ weight data (level of significance 0.05 and 0.01).
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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
Description (incidence and severity):
no toxicological significance
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
on thymus (females)
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
on thymus (males and females)
Histopathological findings: neoplastic:
not examined
Details on results:
MORTALITY:
There were no premature deaths.

CLINICAL SIGNS:
There were no test item-related clinical signs.
Incidental findings included alopecia, thinning of hair, chromorhynorrhea, eye opacity, reflux at dosing and scab, observed in isolated animals among all groups.

BODY WEIGHT (GAIN):
There were no effects on mean body weights or mean body weight gains of toxicological significance.
There was an incidental higher mean body weight gain in the females treated at 2.5 mg/kg/day (+34% vs. controls).

FOOD CONSUMPTION:
There were no effects on mean food consumption.

NEUROBEHAVIOURAL EXAMINATION:
There were no test item-related effects on Functional Observation Battery.
There were no toxicologically significant effects on motor activity. One female at 0.5 mg/kg/day had a low motor activity when compared with the other animals, but this was isolated and no hypoactivity was recorded in clinical sign for this animal. This finding was considered to be fortuitous.

HAEMATOLOGY:
Slightly statistically lower mean hemoglobin concentration and hematocrit (or packed cell volume) were noted in the 10 mg/kg/day females. These findings were ascribed to the test item-treatment. There were no effects in males.

CLINICAL CHEMISTRY:
The differences were considered of no toxicological significance (not dose related, not biologically relevant and/or not associated with other findings).

URINALYSIS:
There were no test item-related urinary abnormalities.

ORGAN WEIGHTS:
There were lower absolute and relative-to-body thymus weights in females treated at 10 mg/kg/day with 4 methylthiosemicarbazide. This organ weight difference correlated with thymus atrophy and was attributed to treatment.
Other weight differences were minimal, not dose-related or of opposing trend between sexes and therefore a relationship to treatment was considered to be unlikely.

GROSS PATHOLOGY:
No macroscopic findings were attributed to treatment with 4-methylthiosemicarbazide.
The gross findings that were observed were of those commonly reported in the rats of this strain and age and therefore a relationship to treatment was excluded.

HISTOPATHOLOGY: NON-NEOPLASTIC:
Thymus
Minimal lymphoid atrophy characterized primarily by a decreased cortex size/cellularity was observed in three out of five females treated at 10 mg/kg/day with 4-methylthiosemicarbazide. This finding correlated with decreased thymus weight in females. This finding was considered to be treatment-related.
There were no test item-related effects on the thymus at 0.5 and 2.5 mg/kg/day.

Spleen
There was minimally increased extramedullary hematopoisesis (primarily erythropoiesis) in the spleen of males and females treated at 10 mg/kg/day. This finding was associated with some changes in blood parameter (hemoglobin, hematocrit) and was attributed to treatment with 4-methylthiosemicarbazide.
There were no test item-related effects on the spleen at 0.5 and 2.5 mg/kg/day.


Bone marrow
There was a trend toward a minimal decrease in adipocyte numbers in the bone marrow of females treated with 4-methylthiosemicarbazide at all dose-levels. This finding was associated with increased erythropoiesis in the spleen in rats treated at 10 mg/kg/day but not in the low- and intermediate-dose. A relationship to treatment could not be entirely excluded at 10 mg/kg/day.

Other microscopic findings were of those commonly reported in the rat of this strain and age and a relationship to treatment was excluded.
Dose descriptor:
NOAEL
Effect level:
2.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified
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 0.5, 2.5 or 10 mg/kg/day.
The No Observed Adverse Effect Level (NOAEL) was considered to be at 2.5 mg/kg/day based on effects observed at 10 mg:kg bw on thymus, spleen and bone marrow.
Executive summary:

The objective of this study was to evaluate the potential toxicity of the test item, 4‑methylthiosemicarbazide, following daily oral administration (gavage) to rats for 4 weeks, according to OECD (No. 407, 3rd October 2008) and EC (No. 440/2008, B7, 30th May 2008) guidelines.

The study was conducted in compliance with the principles of Good Laboratory Practice Regulations.

Methods 

Three groups of five male and five female Sprague-Dawley rats received the test item, 4‑methylthiosemicarbazide, by daily oral administration for 4 weeks at dose‑levels of 0.5, 2.5 or 10 mg/kg/day. The test item was administered as a solution in the vehicle (drinking water treated by reverse osmosis) 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 animals were checked at least twice daily during the dosing period for mortality and morbidity and at least once daily for clinical signs. In addition, detailed clinical examinations were performed once weekly. Body weight was recorded once before the beginning of the treatment period, and then once a week during the study as food consumption.

Towards the end of the dosing period, a Functional Observation Battery including motor activity measurement and hematology, blood biochemistry and urinalysis were performed on all animals. Blood was also taken for a possible further thyroid hormone investigation, but no analysis was performed.

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 and on spleen, bone marrow and thymus from the low- and mid-dose animals, sacrificed at the end of the treatment period.

 

Results

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

 

There were no premature deaths and no test item-related clinical signs or effects on Functional Observation Battery and motor activity. There were no toxicologically relevant effects on mean food consumption, mean body weight or blood biochemistry parameters and no test item-related urinary abnormalities. The only relevant findings were low mean hemoglobin concentration (13.5 vs. 14.6 g/dL in controls, p < 0.01) and hematocrit (0.40 vs. 0.43 L/L in controls, p < 0.05) in females at 10 mg/kg/day. These minimal differences were ascribed to the test item treatment but were considered as non adverse. There were no effects in hematology parameters of males.

At pathological examination, females treated at 10 mg/kg/day had a low mean absolute and relative‑to‑body thymus weight (about -25% vs controls), correlating with minimal thymus atrophy noted in 3/5 females. Microscopic examination also revealed at 10 mg/kg/day an increased extramedullary hematopoiesis in the spleen of both sexes (10/10 animals, vs. 3/10 in controls). These findings were ascribed to the test item treatment but were considered as non adverse. Minimal decrease in adipocyte numbers was also noted in the bone marrow of females at all dose-levels; a relationship to treatment could not be entirely excluded at 10 mg/kg/day as associated with effects seen in the spleen.

Conclusion

 

The test item, 4‑methylthiosemicarbazide, was administered daily for 4 weeks by the oral route to male and female Sprague-Dawley rats at dose-levels of 0.5, 2.5 or 10 mg/kg/day.

 

At 10 mg/kg/day, minimal lymphoid atrophy in thymus was observed in 3/5 females and correlated with decreased thymus weight noted in females. There was also a minimally increased extramedullary hematopoiesis in the spleen of males and females. Moreover there was a trend towards a minimal decrease in adipocyte numbers in the bone marrow of females treated at all dose-levels; a relationship to treatment could not be entirely excluded at 10 mg/kg/day. These effects in the spleen and bone marrow at 10 mg/kg/day were in line with the slightly lower mean hemoglobin concentration and hematocrit noted in females at this dose level and may be suggestive of a slight regenerative anemia.

 

The No Observed Adverse Effect Level (NOAEL) was considered to be at 2.5 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
2.5 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
This 4-week repeated toxicity study is a reliable study performing with OECD guideline and GLP compliance.

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

Repeated dose toxicity: oral - Systemic effects

The objective of study (Bentz 2012) was to evaluate the potential toxicity of the test item, 4‑methylthiosemicarbazide, following daily oral administration to rats for 4 weeks, according to OECD 407 and EC B7 guidelines. Three groups of five male and five female Sprague-Dawley rats received the test item by daily oral administration for 4 weeks at dose‑levels of 0.5, 2.5 or 10 mg/kg/day. The test item was administered as a solution in the vehicle (drinking water treated by reverse osmosis) 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.

There were no premature deaths and no test item-related clinical signs or effects on Functional Observation Battery and motor activity. There were no toxicologically relevant effects on mean food consumption, mean body weight or blood biochemistry parameters and no test item-related urinary abnormalities. The only relevant findings were low mean hemoglobin concentration (13.5 vs. 14.6 g/dL in controls, p < 0.01) and hematocrit (0.40vs. 0.43 L/L in controls, p < 0.05) in females at 10 mg/kg/day. These minimal differences were ascribed to the test item treatment but were considered as non adverse. There were no effects in hematology parameters of males.

At pathological examination, females treated at 10 mg/kg/day had a low mean absolute and relative‑to‑body thymus weight (about -25%vscontrols), correlating with minimal thymus atrophy noted in 3/5 females. Microscopic examination also revealed at 10 mg/kg/day an increased extramedullary hematopoiesis in the spleen of both sexes (10/10 animals,vs.3/10 in controls). These findings were ascribed to the test item treatment but were considered as non adverse. Minimal decrease in adipocyte numbers was also noted in the bone marrow of females at all dose-levels; a relationship to treatment could not be entirely excluded at 10 mg/kg/day as associated with effects seen in the spleen.

To conclude, at 10 mg/kg/day, minimal lymphoid atrophy in thymus was observed in 3/5 females and correlated with decreased thymus weight noted in females. There was also a minimally increased extramedullary hematopoiesis in the spleen of males and females. Moreover there was a trend towards a minimal decrease in adipocyte numbers in the bone marrow of females treated at all dose-levels; a relationship to treatment could not be entirely excluded at 10 mg/kg/day. These effects in the spleen and bone marrow at 10 mg/kg/day were in line with the slightly lower mean hemoglobin concentration and hematocrit noted in females at this dose level and may be suggestive of a slight regenerative anemia.

Based on these results, the No Observed Adverse Effect Level (NOAEL) was considered to be at 2.5 mg/kg/day in rats.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only one study is available for this endpoint.

Justification for classification or non-classification

In the 28-day repeated toxicity study, 4‑methylthiosemicarbazide, was administered daily for 4 weeks by the oral route to male and female Sprague-Dawley rats at dose-levels of 0.5, 2.5 or 10 mg/kg/day. At 10 mg/kg/day, minimal lymphoid atrophy in thymus was observed in 3/5 females and correlated with decreased thymus weight noted in females. There was also a minimally increased extramedullary hematopoiesis in the spleen of males and females. Moreover there was a trend towards a minimal decrease in adipocyte numbers in the bone marrow of females treated at all dose-levels; a relationship to treatment could not be entirely excluded at 10 mg/kg/day. These effects in the spleen and bone marrow at 10 mg/kg/day were in line with the slightly lower mean hemoglobin concentration and hematocrit noted in females at this dose level and may be suggestive of a slight regenerative anemia. Based on these results, the No Observed Adverse Effect Level (NOAEL) was considered to be at 2.5 mg/kg/day. The targets organs were thymus and bone marrow. The spleen was not considered to be a primary target organ, indeed the adverse effects observed on spleen were considered to be adaptive to the effects observed on bone marrow.

Proposed self-classification

- Regulation (EC) No 1272/2008: STOT RE 1 (Thymus and Bone marrow)

Justification:According to the ECHA Guidance on the Application of the CLP Criteria, the Classification in Category 1 is applicable, when significant toxic effects observed in the 28-day repeated-dose study conducted in experimental animals are seen to occur at or below the value of 30 mg/kg bw/d (in rat, by oral route). In the 28 -day study, the LOAEL was 10 mg/kg/d.

 

- Directive 67/548/EEC: R48/25

Justification: The NOAEL is below than 5 mg/kg/d (oral route).