4-methylthiosemicarbazide

Repeated dose toxicity: oral

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

USA EPA Health Effects Test Guideline OPPTS 870.3050, July 2000

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

Reference substance name:

4-methylthiosemicarbazide

EC Number:

229-563-2

EC Name:

4-methylthiosemicarbazide

Cas Number:

6610-29-3

Molecular formula:

C2H7N3S

IUPAC Name:

1-amino-3-methylthiourea

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material: 4-Methylthiosemicarbazide
- Physical state: white powder
- Lot/batch No.: 11012441081
- Analytical purity: 99.74%
- Expiry date: 06 January 2013
- Storage conditions: at room temperature and protected from light and humidity.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system 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.

Administration / exposure

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

Doses / concentrations

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)

Examinations

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).

Results and discussion

Results of examinations

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.

Effect levels

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'

Target system / organ toxicity

Critical effects observed:

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 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, 3^rd October 2008) and EC (No. 440/2008, B7, 30^th 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.