Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: other: chromosome aberration and aneuploidy
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 November 2011 - 24 January 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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: 4-Methylthiosemicarbazide
- Physical state: white powder
- Lot/batch No.: 11012441081
- Molecular weight: 105.16 g/mol
- 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 and environmental conditions:
TEST ANIMALS
- Source: breeder: Charles River Laboratories France, L'Arbresle, France
- Age at study initiation: approximately 6 weeks old on the day of treatment
- Mean body weight at study initiation: at the commencement of the cytogenetic study, the mean body weight was 189 g for males (ranging from 178 g to 208 g) and 146 g for females (ranging from 135 g to 161 g)
- Fasting period before study: no
- Housing: the animals were housed by group from three to five in polycarbonate cages
- 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 5 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: 06 December 2011 to 05 January 2012.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: drinking water
- Concentration of test material in vehicle: for the main test, three dosage forms were prepared at 0.25, 0.5 and 1 mg/mL
- Justification for choice of solvent/vehicle: according to available solubility data, the vehicle was drinking water as used in a 4-week toxicity study in
rats.
- Amount of vehicle (if gavage or dermal): 10 mL/kg.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved in the vehicle and the preparations were homogenized using a magnetic stirrer.
Dosage forms were prepared within the 4 hours before use, and then kept at room temperature and protected from light until use. The dosage forms were delivered to the study room in brown flasks, at room temperature.
For the main test, using a treatment volume of 10 mL/kg, the target dose-levels were 2.5, 5 and 10 mg/kg/day. Therefore, three dosage forms were prepared at 0.25, 0.5 and 1 mg/mL.
Duration of treatment / exposure:
Two treatments separated by 24 hours.
Frequency of treatment:
One treatment per day.
Post exposure period:
Sacrifice: 24 hours after the last treatment
Doses / concentrations
Remarks:
Doses / Concentrations:
2.5, 5 and 10 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
5 males and 5 females at 2.5 and 5mg/kg/day, as well as for the vehicle and positive control groups.
8 males and 8 females at 10 mg/kg/day.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: oral
- Doses / concentrations: 15 mL/kg.

Examinations

Tissues and cell types examined:
Bone marrow: polychromatic (PE) and normochromatic (NE) erythrocytes.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Since toxic effects were observed in the preliminary dose range-finding tests, the choice of the top dose-level was based on the level of toxicity, such that a higher dose-level was expected to induce lethality.

SAMPLING TIMES:
At sacrifice, 24 h after the last treatment.

DETAILS OF SLIDE PREPARATION:
After sacrifice, the femurs were removed and bone marrow was flushed and suspended in fetal calf serum. The separation of anucleated erythrocytic
cells from other myeloic cells was carried using a cellulose column. This elution step enables the production of slides containing only polychromatic
and normochromatic erythrocytes without any nucleated cells or mast cell granules. After centrifugation of the eluate containing the cells, the
supernatant was removed and the cells in the sediment were resuspended. A drop of this cell suspension was placed and spread on a slide. At least
two slides were prepared for each animal. The slides were air-dried and stained with Giemsa and then coded for "blind" scoring.

METHOD OF ANALYSIS:
For each animal, the number of the Micronucleated Polychromatic Erythrocytes (MPE) was counted in 2000 Polychromatic Erythrocytes; the
Polychromatic (PE) and Normochromatic (NE) Erythrocyte ratio was established by scoring a total of 1000 Erythrocytes (PE + NE).
Evaluation criteria:
For a result to be considered positive, there must be: a statistically significant increase in the frequency of MPE when compared to the vehicle control group. Reference to historical data or other considerations of biological relevance may be taken into account in the evaluation of data obtained.
Statistics:
The analyses of MPE/1000PE and PE/NE ratios were performed with SAS software (version 9.2, SAS Institute Inc.).

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
In order to select the top dose-level for the cytogenetic study, 10 mg/kg/day was first administered twice, to three males and three females.
As no deaths and no clinical signs were noted during the observation period, a second preliminary test was performed at a higher dose-level
of 50 mg/kg/day.
At this dose-level, one male and one female were found dead two hours after the first treatment. The female had severe clinical signs prior to its death animals (ventral recumbancy, clonic convulsions, ptyalism, abdominal breathing). Signs of poor clinical condition (abnormal vocalization, hypoactivity, clonic convulsions, ptyalism, abdominal breathing and/or tremors) were observed in the surviving animals. Based on the mortality and the clinical signs observed, no second treatment was performed on the surviving animals which were sacrificed for ethical reasons before the end of the observation period.
A third preliminary test was performed at the lower dose-level of 20 mg/kg/day. At this dose level, 2/3 males were found dead on day 2 before the second administration. No clinical signs were noted in the surviving animals. Based on the mortality observed, no second treatment was performed on the surviving animals which were sacrificed for ethical reasons before the end of the observation period.

The top dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines. Since toxic effects were observed, the choice of the top dose-level was based on the level of toxicity, such that a higher dose-level was expected to induce lethality.

Consequently, 10 mg/kg/day was selected as the top dose-level for the main test. The two other selected dose-levels were 2.5 and 5 mg/kg/day.

- Clinical signs of toxicity in test animals:
Neither mortality nor clinical signs were observed in the animals of either sex from the positive control and from the vehicle control groups.
No mortality and no clinical signs were observed in the animals at any tested dose-levels.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test item did not induce damage to the chromosomes or the mitotic apparatus of rat bone marrow cells after two oral administrations,
at a 24-hour interval, at the dose-levels of 2.5, 5 and 10 mg/kg/day.
Executive summary:

The objective of this study was to evaluate the potential of the test item, 4‑Methylthiosemicarbazide, to damage to the chromosomes or the mitotic apparatus in bone marrow cells of rats.

 

The study was performed according to the international guidelines (OECD guideline No. 474 and Council Regulation No. 440/2008 of 30 May 2008, Annex, Part B.12) and in compliance with the principles of Good Laboratory Practice. 

 

Methods

Preliminary toxicity tests were performed to define the dose-levels to be used for the cytogenetic study.

In the main study, three groups of five male and five female Sprague-Dawley rats received two oral treatments of 4-Methylthiosemicarbazide at dose-levels of 2.5, 5 and 10 mg/kg/day, at a 24-hour interval. For the high-dose group only, three supplementary males and three supplementary females were also treated with the test item in case of mortality.

One group of five males and five females received the vehicle (drinking water treated by reverse osmosis) under the same experimental conditions, and acted as control group.

One group of five males and five females received the positive control (cyclophosphamide) once by oral route at the dose-level of 15 mg/kg/day.

The animals of the treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared.

For each animal, the number of the Micronucleated Polychromatic Erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The Polychromatic (PE) and Normochromatic (NE) Erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

 

Results

The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with our historical data. Cyclophosphamide induced a highly significant increase (p < 0.001) in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered to be valid.

In all groups treated with 4-Methylthiosemicarbazide, the mean values of the MPE and the PE/NE ratio weresimilar to those of their respective vehicle controls, and nostatistically significant differences were noted.Therefore, the criteria for a positive response were not met.

 

Conclusion

The test item did not induce damage to the chromosomes or the mitotic apparatus of rat bone marrow cells after two oral administrations,

at a 24-hour interval, at the dose-levels of 2.5, 5 and 10 mg/kg/day.