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Toxicological information

Genetic toxicity: in vivo

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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:
18 April 2012 - 14 June 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)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material: 1,3-Diphenyl-2-thiourea, DPTU
- Physical state: white powder
- Lot/batch No.: 1012001047
- Analytical purity: 99.4%
- Expiry date: 12 January 2013
- Storage conditions: protected from moisture and heat, at room temperature.

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 212 g for males (ranging from 199 g to 232 g) and 165 g for females (ranging from 145 g to 178 g)
- Fasting period before study: no
- Housing: The animals were housed by two or three, by sex and group, 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.

IN-LIFE DATES: 09 May 2012 to 14 June 2012.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: corn oil
- Amount of vehicle (if gavage or dermal): 10 mL/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was given in the vehicle. The test item was ground to a fine powder using a mortar and pestle, suspended in the vehicle and then homogenized using a magnetic stirrer. The preparations were maintained under agitation throughout the treatment period. Dose-formulations were prepared within 9 days before use, and then kept at room temperature and protected from light until use (according to the results of the homogeneity and stability study. The dose-formulations were delivered to the study room in brown flasks, at room temperature.

Three dose-formulations were prepared for the main test, at concentrations of 50, 100 and 200 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:
500, 1000 and 2000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
5 males and 5 females at 500 and 1000 mg/kg/day.
7 males and 8 females at 2000 mg/kg/day.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: oral
- Doses / concentrations: 10 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:
In order to determine the high dose-level for use in the cytogenetic study and depending upon the amount of information supplied by the Sponsor, a preliminary test was performed on a group of six animals (three males and three females). According to the available information, the starting dose-level was 2000 mg/kg/day (group 1).

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. 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:
no

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
- Dose range: 2000 mg/kg (2 times)
- Clinical signs of toxicity in test animals: No mortalities and no clinical signs were observed at any of the tested dose-levels during the study.

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, 24-hour apart, at the dose-levels of 500, 1000 and 2000 mg/kg/day.
Executive summary:

The objective of this study was to evaluate the potential of the test item to induce damage to the chromosomes or the mitotic apparatus in rat bone marrow cells.

The study was performed according to the international guidelines (OECD 474 and Commission Directive No. B12) and in compliance with the Principles of Good Laboratory Practice. 

 

Methods

A preliminary toxicity test was 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 1,3-Diphenyl-2-thiourea at dose-levels of 500, 1000 and 2000 mg/kg/day, at a 24-hour interval. For the high-dose group only, two 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 (corn oil) under the same experimental conditions, and acted as control group.

One group of five males and five females received the positive control test item (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

According to the criteria specified in the international guidelines, since no toxic effects were observed at 2000 mg/kg/day in the preliminary test, this dose-level was selected as the top dose‑level for the main test. The two other selected dose-levels were 500 and 1000 mg/kg/day.

 

No mortalities and no clinical signs were observed at any of the tested dose-levels during the study.

 

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 significant increase (p < 0.001 males and p < 0.05 females) in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered to be valid.

 

The test item did not induce any noteworthy decrease in the PE/NE ratios when compared to the vehicle control group.

 

The mean values of MPE in the test item-treated groups were found equivalent to those of the vehicle group. These results met the criteria of a negative response.

Conclusion

The test item did not induce damage to the chromosomes or the mitotic apparatus of rat bone marrow cells after two oral administrations, 24-hour apart, at the dose-levels of 500, 1000 and 2000 mg/kg/day.