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Genetic toxicity in vitro

Description of key information

Several in vitro studies are available to evaluate the mutagenicity of DPTU. Ames test showed negative results with and without metabolic activation. The mouse lymphoma assay (OECD 476) showed positive response with metabolic activation, with a majority of small colonies which suggested a possible clastogenic effect.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 January 2012 - 23 April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.
Test concentrations with justification for top dose:
Experiments without S9 mix
The selected treatment-levels were 78.13, 156.3, 312.5, 625, 1250 and 2500 µg/plate for the first and second experiments.

Experiments with S9 mix
.           78.13, 156.3, 312.5, 625, 1250 and 2500 µg/plate for the first and second experiments,
.           312.5, 625, 937.5, 1250, 1875 and 2500 µg/plate for the third and fourth experiments.
Vehicle / solvent:
- Vehicle used: dimethylsulfoxide
- Justification for choice: test item is soluble at 100 mg/mL in the vehicle and using a treatment volume of 50 µL/plate, the highest recommended dose-level of 5000 µg/plate is achievable.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, 9-aminoacridine, 2-nitrofluorene, mitomycin C (-S9 mix); 2-anthramine, benzo(a)pyrene (+S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar

The first experiment (with and without S9 mix) and the second experiment without S9 mix were performed according to the direct plate incorporation method, contrary to the second, third and fourth experiments with S9 mix, which were performed according to the pre-incubation method (60 minutes, 37°C).
Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to six dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.
The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.
DURATION
- Preincubation period: 60 minutes at 37°C.

DETERMINATION OF CYTOTOXICITY
- Method: decrease in number of revertant colonies and/or thinning of the bacterial lawn
Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account.
Statistics:
no
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The number of revertants for the vehicle and positive controls met the acceptance criteria. The study was therefore considered to be valid.
Since the test item was found poorly soluble in the preliminary test, the choice of the highest dose-level was based on the level of precipitate, according to the criteria specified in the international guidelines.

Experiments without S9 mix
The selected treatment-levels were 78.13, 156.3, 312.5, 625, 1250 and 2500 µg/plate for the first and second experiments.
A moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels >= 1250 µg/plate.
In the first experiment, a moderate toxicity (thinning of the bacterial lawn) was noted at dose-levels of 1250 µg/plate in the strains TA 1535 and TA 1537, and at 2500 µg/plate in the TA 100 strain.
In the second experiment, a moderate toxicity (thinning of the bacterial lawn) was noted at 2500 µg/plate in the TA 98 strain.
No noteworthy toxicity was noted towards the strain TA 102 in either experiment.
The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains.
 
Experiments with S9 mix:
In the first experiment performed using the direct plate incorporation method, a moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels >= 1250 µg/plate.
Using the pre-incubation method, a moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels >=1250 µg/plate, except for the strains TA 100 and TA 1537 in the second experiment where a moderate precipitate was observed at 1250 µg/plate only. In the fourth experiment, a strong toxicity (decrease in the number of revertants) was noted at 2500 µg/plate towards the strain TA 1537.
No noteworthy toxicity was noted towards the other strains used.
 
Noteworthy increases in the number of revertants were noted at 2500 µg/plate in the strains TA 1535, TA 1537 and TA 100 in the second experiment performed with S9 mix. These increases exceeded the threshold of 2-fold the vehicle control (2.6-fold the vehicle control for the strain TA 100) and of 3-fold the vehicle control (up to 30.3-fold the vehicle control for the strains TA 1535 and TA 1537). Two additional experiments were performed using the same experimental conditions (pre-incubation method) and a closer range of dose-levels to check the reproducibility and therefore the reliability of these increases. In these experiments, no increases in the number of revertants were noted at any of the dose-levels tested. Thus, the increases noted in the second experiment were not reproducible in two additional independent experiments. Consequently, they were not considered as biologically relevant.
Conclusions:
The test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium, in the presence or in the absence of a rat metabolizing system.
Executive summary:

The objective of this study was to evaluate the potential of the test item to induce reverse mutation in Salmonella typhimurium.

The study was performed according to the international guidelines (OECD No. 471 and Council Regulation (EC) No. 440/2008 of 30 May 2008, Part B13/14 p. 248) and in compliance with the principles of Good Laboratory Practice.

 

Methods

The first experiment (with and without S9 mix) and the second experiment without S9 mix were performed according to the direct plate incorporation method, contrary to the second, third and fourth experiments with S9 mix, which were performed according to the pre-incubation method (60 minutes, 37°C).

Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to six dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

Results

The test item was dissolved in dimethylsulfoxide (DMSO).

The number of revertants for the vehicle and positive controls met the acceptance criteria. The study was therefore considered to be valid.

Since the test item was found poorly soluble in the preliminary test, the choice of the highest dose-level was based on the level of precipitate, according to the criteria specified in the international guidelines.


Experiments without S9 mix

The selected treatment-levels were 78.13, 156.3, 312.5, 625, 1250 and 2500 µg/plate for the first and second experiments.

A moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels >= 1250 µg/plate.

In the first experiment, a moderate toxicity (thinning of the bacterial lawn) was noted at dose-levels of 1250 µg/plate in the strains TA 1535 and TA 1537, and at 2500 µg/plate in the TA 100 strain.

In the second experiment, a moderate toxicity (thinning of the bacterial lawn) was noted at 2500 µg/plate in the TA 98 strain.

No noteworthy toxicity was noted towards the strain TA 102 in either experiment.

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains.

 

Experiments with S9 mix

In the first experiment performed using the direct plate incorporation method, a moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels >= 1250 µg/plate.

Using the pre-incubation method, a moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels >=1250 µg/plate, except for the strains TA 100 and TA 1537 in the second experiment where a moderate precipitate was observed at 1250 µg/plate only. In the fourth experiment, a strong toxicity (decrease in the number of revertants) was noted at 2500 µg/plate towards the strain TA 1537.

No noteworthy toxicity was noted towards the other strains used.

 

Noteworthy increases in the number of revertants were noted at 2500 µg/plate in the strains TA 1535, TA 1537 and TA 100 in the second experiment performed with S9 mix. These increases exceeded the threshold of 2-fold the vehicle control (2.6-fold the vehicle control for the strain TA 100) and of 3-fold the vehicle control (up to 30.3-fold the vehicle control for the strains TA 1535 and TA 1537). Two additional experiments were performed using the same experimental conditions (pre-incubation method) and a closer range of dose-levels to check the reproducibility and therefore the reliability of these increases. In these experiments, no increases in the number of revertants were noted at any of the dose-levels tested. Thus, the increases noted in the second experiment were not reproducible in two additional independent experiments. Consequently, they were not considered as biologically relevant.

 

Conclusion

The test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium, in the presence or in the absence of a rat metabolizing system.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
ation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 January 2012 - 28 February 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine Kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium
pyruvate (200 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
Without S9 :
6.25, 12.5, 25, 50, 100 and 200 µg/mL for the first experiment (3-hour treatment),
1.56, 3.13, 6.25, 12.5, 25 and 50 µg/mL for the second experiment (24-hour treatment).

With S9 :
3.13, 6.25, 12.5, 25, 50 and 100 µg/mL for the first experiment (3-hour treatment),
1.56, 3.13, 6.25, 12.5, 18.8, 25 and 50 µg/mL for the second experiment (3-hour treatment).
Vehicle / solvent:
- Vehicle used: dimethylsulfoxide (DMSO)
- Justification for choice: test item was soluble in the vehicle at 100 mg/mL.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: methylmethanesulfonate (-S9 mix); cyclophosphamide (+S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
Cultures of 20 mL at 5 x 105cells/mL (3-hour treatment) or cultures of 50 mL at 2 x 105cells/mL (24-hour treatment) were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% (3-hour treatment) or 10% (24-hour treatment) in a, 5% CO2 humidified incubator. For the 24-hour treatment, flasks were gently shaken at least once.

DURATION
- Exposure duration: 3 and 24 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 11-12 days

SELECTION AGENT (mutation assays): trifluorothymidine

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth.
Evaluation criteria:
Positive result defined as:
- At least at one dose-level the mutation frequency minus the mutation frequency of the vehicle control (IMF) equals or exceeds the global evaluation factor (GEF) of 126 E-6
- A dose-related trend is demonstrated by a statistically significant trend test
- Unless clearly positive, the reproducibility should be confirmed

Negative results defined as:
- No evidence of mutagenicity at concentrations inducing moderate cytotoxicity (10% < Adj. RTG <20%), or
- If there no culture has 10% < Adj. RTG <20%:
¿ at least one negative data point with 20% < Adj. RTG <25% + negative data from 20% < Adj. RTG <100%, or
¿ at least one negative data point with 1% < Adj. RTG <10% + negative data from 25% < Adj. RTG <100%
Statistics:
no
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
With a few exceptions which were not considered to have a biological impact on the validity of the study, the Cloning Efficiencies (CE2), the Suspension Growths (SG) and the mutation frequencies of the vehicle and positive controls were considered as specified in the acceptance criteria. The study was therefore considered to be valid.
Since the test item was toxic in the preliminary test, the choice of the highest dose-level for the main test was based on the level of toxicity, according to the criteria specified in the international guidelines (decrease in Adj. RTG).
 
Experiments without S9 mix
The selected dose-levels were as follows:
. 6.25, 12.5, 25, 50, 100 and 200 µg/mL for the first experiment (3-hour treatment),
. 1.56, 3.13, 6.25, 12.5, 25 and 50 µg/mL for the second experiment (24-hour treatment).
At the end of the 3-hour treatment, a precipitate was noted at dose-level of 200 µg/mL.
 
Cytotoxicity
Following the 3-hour treatment, a slight to severe toxicity was induced at dose-levels comprise between 25 and 100 µg/mL, as shown by a 34-98% decrease in the Adj. RTG. At 200 µg/mL, no evaluation of the decrease in the Adj. RTG could be performed since no cell was viable after the expression period.
Following the 24-hour treatment, a slight to severe toxicity was induced at dose-levels = 12.5 µg/mL, as shown by a 30-100% decrease in the Adj. RTG.

Mutagenicity
Following the 3-hour treatment, a slight increase in the mutation frequency was observed at dose-levels = 50 µg/mL. This increase did not exceed the GEF even at the too cytotoxic dose-level of 100 µg/mL (inducing a 98% decrease in the Adj. RTG). Thus this result did not meet the criteria of a positive response.
 
Following the 24-hour treatment, no increase in the mutation frequency was observed up to (and even above) the dose-level of 25 µg/mL, inducing a 90% decrease in the Adj. RTG. Thus this result did not meet the criteria of a positive response.

Experiments with S9 mix
The selected dose-levels were as follows:
. 3.13, 6.25, 12.5, 25, 50 and 100 µg/mL for the first experiment (3-hour treatment),
. 1.56, 3.13, 6.25, 12.5, 18.8, 25 and 50 µg/mL for the second experiment (3-hour treatment).
 
At the end of the first experiment, no precipitate was noted at any dose-levels.
 
Cytotoxicity
In the first and second experiments, a marked to severe toxicity was induced at dose-levels = 12.5 µg/mL, as shown by a 68-100% decrease in the Adj. RTG.
 
Mutagenicity
In the first experiment, dose-related increases in the mutation frequency were observed at dose-levels = 6.25 µg/mL. These increases exceeded the global evaluation factor of +126 x 10-6at the dose-level of 12.5 µg/mL, inducing an acceptable level of cytotoxicity (68% decrease in the Adj. RTG).Thus this result met the criteria of a positive response.
In the second experiment, dose-related increases in the mutation frequency were observed at dose-levels = 6.25 µg/mL. These increases exceeded the GEF at the dose-levels of 12.5 and 18.8 µg/mL, inducing acceptable levels of cytotoxicity (76 and 90% decrease in the Adj. RTG, respectively). Thus this result met the criteria of a positive response.
Compared to the vehicle control, the mutation frequencies in the second experiment were increased of up to 82 x 10-6 and 130 x 10-6, for the large and small colonies respectively at 12.5 µg/mL, and 128 x 10-6and 220 x 10-6, for the large and small colonies respectively at 18.8 µg/mL. This might indicate that the test item induced chromosome damages as well as point mutations.
Conclusions:
The test item showed a mutagenic activity in the mouse lymphoma assay, in the presence of a rat metabolizing system, whereas it did not in the absence of metabolic activation. Moreover, a high number of small colonies were observed in the positive second experiment, this might indicate that the test item induced chromosome damages as well as point mutations.
Executive summary:

The objective of this study was to evaluate the potential of the test item to induce mutations at the TK (Thymidine Kinase) locus in L5178Y TK+/-mouse lymphoma cells. The study was performed according to international guidelines (OECD No. 476 and Council Regulation No. 440/2008 of 30 May 2008) and in compliance with the principles of Good Laboratory Practice.

After a preliminary toxicity test, 1,3-Diphenyl-2-thiourea, was tested in two independent experiments, with and without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Cytotoxicity wasmeasured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) andCloning Efficiency following the expression time (CE2). The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.

The test item was dissolved in dimethylsulfoxide (DMSO).

 

With a few exceptions which were not considered to have a biological impact on the validity of the study, the Cloning Efficiencies (CE2), the Suspension Growths (SG) and the mutation frequencies of the vehicle and positive controls were considered as specified in the acceptance criteria. The study was therefore considered to be valid.

Since the test item was toxic in the preliminary test, the choice of the highest dose-level for the main test was based on the level of toxicity, according to the criteria specified in the international guidelines (decrease in Adj. RTG).

 

Experiments without S9 mix

At the end of the 3-hour treatment (first experiment), a precipitate was noted at dose-level of 200 µg/mL.

Following the 3-hour treatment, a slight to severe toxicity was induced at dose-levels comprise between 25 and 100 µg/mL, as shown by a 34-98% decrease in the Adj. RTG. At 200 µg/mL, no evaluation of the decrease in the Adj. RTG could be performed since no cell was viable after the expression period. Following the 24-hour treatment (second experiment), a slight to severe toxicity was induced at dose-levels = 12.5 µg/mL, as shown by a 30-100% decrease in the Adj. RTG.

Following the 3-hour treatment, a slight increase in the mutation frequency was observed at dose-levels = 50 µg/mL. This increase did not exceed the GEF even at the too cytotoxic dose-level of 100 µg/mL (inducing a 98% decrease in the Adj. RTG). Thus this result did not meet the criteria of a positive response. Following the 24-hour treatment, no increase in the mutation frequency was observed up to (and even above) the dose-level of 25 µg/mL, inducing a 90% decrease in the Adj. RTG.

Thus this result (without S9) did not meet the criteria of a positive response.

Experiments with S9 mix

At the end of the first experiment, no precipitate was noted at any dose-levels.

In the first and second experiments, a marked to severe toxicity was induced at dose-levels = 12.5 µg/mL, as shown by a 68-100% decrease in the Adj. RTG.

In the first experiment, dose-related increases in the mutation frequency were observed at dose-levels = 6.25 µg/mL. These increases exceeded the global evaluation factor of +126 x 10^-6 at the dose-level of 12.5 µg/mL, inducing an acceptable level of cytotoxicity (68% decrease in the Adj. RTG).Thus this result met the criteria of a positive response.

In the second experiment, dose-related increases in the mutation frequency were observed at dose-levels = 6.25 µg/mL. These increases exceeded the GEF at the dose-levels of 12.5 and 18.8 µg/mL, inducing acceptable levels of cytotoxicity (76 and 90% decrease in the Adj. RTG, respectively). Thus this result met the criteria of a positive response.

Compared to the vehicle control, the mutation frequencies in the second experiment were increased of up to 82 x 10^-6 and 130 x 10^-6, for the large and small colonies respectively at 12.5 µg/mL, and 128 x 10^-6 and 220 x 10^-6, for the large and small colonies respectively at 18.8 µg/mL. This might indicate that the test item induced chromosome damages as well as point mutations.

 

To conclude the test item showed a mutagenic activity in the mouse lymphoma assay, in the presence of metabolizing system, whereas it did not in the absence of metabolic activation. Moreover, a high number of small colonies were observed in the positive second experiment, this might indicate that the test item induced chromosome damages as well as point mutations.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

An in vivo micronucleus test was performed by oral route in rat: no clastogenic effect was observed in this in vivo study.

A Comet assay is proposed to the authorities to verify if the positive MLA TK test is relevant or not.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
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:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay
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 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.
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
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
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.
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
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.
Conclusions:
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Bacterial reverse mutation test : Sarlang (2012)


The objective of this study (OECD 471) was to evaluate the potential of the test item to induce reverse mutation inSalmonella typhimurium.


The first experiment (with and without S9 mix) and the second experiment without S9 mix were performed according to the direct plate incorporation method, contrary to the second, third and fourth experiments with S9 mix, which were performed according to the pre-incubation method (60 minutes, 37°C). Five strains of bacteriaSalmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to six dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. The test item was dissolved in dimethylsulfoxide (DMSO).


The number of revertants for the vehicle and positive controls met the acceptance criteria. The study was therefore considered to be valid.


Since the test item was found poorly soluble in the preliminary test, the choice of the highest dose-level was based on the level of precipitate, according to the criteria specified in the international guidelines.


Experiments without S9 mix: The selected treatment-levels were 78.13, 156.3, 312.5, 625, 1250 and 2500 µg/plate for the first and second experiments. A moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels >= 1250 µg/plate.


In the first experiment, a moderate toxicity (thinning of the bacterial lawn) was noted at dose-levels of 1250 µg/plate in the strains TA 1535 and TA 1537, and at 2500 µg/plate in the TA 100 strain.


In the second experiment, a moderate toxicity (thinning of the bacterial lawn) was noted at 2500 µg/plate in the TA 98 strain.


No noteworthy toxicity was noted towards the strain TA 102 in either experiment.


The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains.


 


Experiments with S9 mix : In the first experiment performed using the direct plate incorporation method, a moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels >= 1250 µg/plate.


Using the pre-incubation method, a moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels >=1250 µg/plate, except for the strains TA 100 and TA 1537 in the second experiment where a moderate precipitate was observed at 1250 µg/plate only. In the fourth experiment, a strong toxicity (decrease in the number of revertants) was noted at 2500 µg/plate towards the strain TA 1537.


No noteworthy toxicity was noted towards the other strains used.


 Noteworthy increases in the number of revertants were noted at 2500 µg/plate in the strains TA 1535, TA 1537 and TA 100 in the second experiment performed with S9 mix. These increases exceeded the threshold of 2-fold the vehicle control (2.6-fold the vehicle control for the strain TA 100) and of 3-fold the vehicle control (up to 30.3-fold the vehicle control for the strains TA 1535 and TA 1537). Two additional experiments were performed using the same experimental conditions (pre-incubation method) and a closer range of dose-levels to check the reproducibility and therefore the reliability of these increases. In these experiments, no increases in the number of revertants were noted at any of the dose-levels tested. Thus, the increases noted in the second experiment were not reproducible in two additional independent experiments. Consequently, they were not considered as biologically relevant.


The test item did not show any mutagenic activity in the bacterial reverse mutation test withSalmonella typhimurium, in the presence or in the absence of a rat metabolizing system.


 


In vitro in mammalian cells gene mutation test (OECD 476) : Sarlang (2012)


The objective of this study (OECD 476) was to evaluate the potential of the test item to induce mutations at the TK (Thymidine Kinase) locus in L5178Y TK+/-mouse lymphoma cells.


After a preliminary toxicity test, 1,3-Diphenyl-2-thiourea, was tested in two independent experiments, with and without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.Cytotoxicity was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE2).The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.


The test item was dissolved in dimethylsulfoxide (DMSO).


With a few exceptions which were not considered to have a biological impact on the validity of the study, the Cloning Efficiencies (CE2), the Suspension Growths (SG) and the mutation frequencies of the vehicle and positive controls were considered as specified in the acceptance criteria. The study was therefore considered to be valid.


Since the test item was toxic in the preliminary test, the choice of the highest dose-level for the main test was based on the level of toxicity, according to the criteria specified in the international guidelines (decrease in Adj. RTG).


At the end of the 3-hour treatment (first experiment without S9), a precipitate was noted at dose-level of 200 µg/mL.


Following the 3-hour treatment (without S9), a slight to severe toxicity was induced at dose-levels comprise between 25 and 100 µg/mL, as shown by a 34-98% decrease in the Adj. RTG. At 200 µg/mL, no evaluation of the decrease in the Adj. RTG could be performed since no cell was viable after the expression period.Following the 24-hour treatment (second experiment without S9), a slight to severe toxicity was induced at dose-levels = 12.5 µg/mL, as shown by a 30-100% decrease in the Adj. RTG.


Following the 3-hour treatment without S9, a slight increase in the mutation frequency was observed at dose-levels = 50 µg/mL. This increase did not exceed the GEF even at the too cytotoxic dose-level of 100 µg/mL (inducing a 98% decrease in the Adj. RTG). Thus this result did not meet the criteria of a positive response.Following the 24-hour treatment without S9, no increase in the mutation frequency was observed up to (and even above) the dose-level of 25 µg/mL, inducing a 90% decrease in the Adj. RTG. Thus this result (without S9) did not meet the criteria of a positive response.


In the first and second experiments with S9, a marked to severe toxicity was induced at dose-levels = 12.5 µg/mL, as shown by a 68-100% decrease in the Adj. RTG.At the end of the first experiment, no precipitate was noted at any dose-levels.


In the first experiment with S9, dose-related increases in the mutation frequency were observed at dose-levels = 6.25 µg/mL. These increases exceeded the global evaluation factor of +126 x 10^-6 at the dose-level of 12.5 µg/mL, inducing an acceptable level of cytotoxicity (68% decrease in the Adj. RTG).Thus this result met the criteria of a positive response.


In the second experiment with S9, dose-related increases in the mutation frequency were observed at dose-levels = 6.25 µg/mL. These increases exceeded the GEF at the dose-levels of 12.5 and 18.8 µg/mL, inducing acceptable levels of cytotoxicity (76 and 90% decrease in the Adj. RTG, respectively). Thus this result met the criteria of a positive response.


Compared to the vehicle control, the mutation frequencies in the second experiment with S9 were increased of up to 82 x 10^-6 and 130 x 10^-6, for the large and small colonies respectively at 12.5 µg/mL, and 128 x 10^-6 and 220 x 10^-6, for the large and small colonies respectively at 18.8 µg/mL. This might indicate that the test item induced chromosome damages as well as point mutations.


To conclude the test item showed a mutagenic activity in the mouse lymphoma assay, in the presence of metabolizing system, whereas it did not in the absence of metabolic activation. Moreover, a high number of small colonies were observed in the positive second experiment, this might indicate that the test item induced chromosome damages as well as point mutations.


 


In vivo micronucleus test : Sire (2012)


The objective of this study (OECD 474) was to evaluate the potential of the test item to induce damage to the chromosomes or the mitotic apparatus in rat bone marrow cells.


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.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).


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.


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.

Justification for classification or non-classification

Based on the available data, no conclusion can be done for mutagenicity according to the Regulation EC 1272/2008 : a new in vivo testing was proposed.