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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three studies were conducted with 1,1 -Dimethylurea in order to evaluate the genetic toxicity the substance:

1,1 -Dimethylurea was tested for mutagenic activity with the "Salmonella typhimurium Reverse Mutation Test" (Ames Test). The study was conducted in accordance with the OECD-guideline 471 and directive 2000/32/EC, part B.13/14.

According to the results obtained in this study, 1,1 -Dimethylurea is non-mutagenic in the Ames test with the strains TA97a, TA98, TA100, TA102 and TA1535 up to 5000 µg/plate, which is the limit concentration for this kind of test.

 

The substance 1,1 -Dimethylurea was tested in an in vitro Mammalian Chromosome Aberration Test using Chinese hamster lung (CHL) cells cultures. Treatments were performed both in the absence and presence of a S9 activation system.

Based on the findings of this study,1,1 -Dimethylurea was concluded to be negative for the induction of structural chromosome aberrations in both non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHL.

 

The test item 1,1 -Dimethylurea was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y according to OECD 490.

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item 1,1-Dimethylurea is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Link to relevant study records

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Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March - October 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
other: The Guidelines for the Testing of Chemicals (Ministry of Environmental Protection of the People’s Republic of China, 2004)
Qualifier:
according to guideline
Guideline:
other: The Guidelines for the hazard Evaluation of New Chemical Substances (Ministry of Environmental Protection of the People’s Republic of China, 2004)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Analytical purity: 99.9 %
- Lot/batch No.: 301601
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
mammalian liver post-mitrochondrial fraction (S9)
Test concentrations with justification for top dose:
Three adequately spaced concentrations of the test substance were used (50, 500, 5000 µg/mL). The highest concentration gave rise to a significant toxic effect but still allowed adequate cell replication to occur. Cells were exposed to the test substance in the presence and absence of S9-mix. The cultures were treated with the test substance preparation in the presence of S9 for 6 hours and in the absence for 6 and 24 hours. At the end of the exposure period, cells were washed free of test substance and cultured for two rounds of replication.
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
see "any other information on materials and methods incl. tables"
Evaluation criteria:
In the following two cases the results of the experimental system would be determined as positive:
(1) The number of chromosome structural aberrations caused by the test substance increases statistically significantly and the increase is concentration-dependent.
(2) The test substance causes a statistically significant increase of structural aberrations and the increase is shown to be repeatable.

Negative results are only stated when cell toxicity was obvious and chromosomal aberrations significantly increased. In this case the evaluation must consider the biological and statistical significance of the finding.
Statistics:
Statistical analysis of the percent aberrant cells was performed using the Chi-square test to evaluate if there were any significant differences between the dose and control groups.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Significant toxic effects in highest concentration, but adequate cell replication still occured
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
see "any other information on results incl. tables"

Preliminary Toxicity Assay

Dose levels for the chromosome aberration assay were selected following the results of a preliminary toxicity test and were based upon a reduction in the mitotic index, relative to the solvent control. Based on Trypan blue counting method, the IC50(IC50: concentration at which the cell inhibition ratio reached 50%) was greater than 5000μg/ml. Since the cytotoxicity of the test substance is relatively low, 5000 ug/ml was adopted as the highest concentration in the definite test. The other two concentration levels were selected to be 500 ug/ml and 50 ug/ml accordingly.

 

Chromosome Aberration Assay

In the absence ofS9- mixandtreatment for 6 h, the cell aberration rate of blank control, solvent control, 50, 500, 5000 ug/ml dose group and the positive control was 0.5 %,0.5 %,1 %,2 %,2.5 %,31.5 %, respectively (see table 2). The mitosis inhibition rates of the three test substance concentrations were6.49%19.12%44.09%, respectively (see table 1).

In the presence ofS9 and treatment for 6 h, the cell aberration rate ofblank control, solvent control,50, 500, 5000 ug/ml dose group and the positive control cell aberration rate was 1 %,0.5 %, 0.5 %,1 %, 1.5 %,27.5 % respectively (see table 2). The mitotic inhibition rates of the test substance concentrations were0.05 %20.31 %42.00 %, respectively (see table 1).

In the absence of S9 and treatment for 24 h, the cell aberration rate of blank control, solvent control, 50, 500, 5000 ug/ml dose group and the positive control cell aberration rate was1 %,1 %,1.5 %,2 %,2.5 %,36 %, respectively (see table 3). The mitosis inhibition rates for the three test substance concentrations were1.17 %34.66 %55.24 % (see table 1).

 

Statistical analysis of the percent aberrant cells was performed using the Chi-square test to evaluate if there are any significant differences between the dose and control groups. When comparing the positive control group with the solvent control group, the differences were statistically significant (P <0.01). Treated groups compared with the solvent control group, showed a difference that was not statistically significant (P>0.05).

Table 1: Results of the mitotic indices.

Treatment

(ug/ml)

-S96h

Mitotic index

 

- S96h

inhibition rate

+ S96h

Mitotic index

 

+ S96h

inhibition rate

- S924h

Mitotic index

 

- S924h

inhibition rate

Solvent control

6.15%

-

6.14%

-

6.00%

-

50

5.40%

6.49%

6.09%

0.05%

5.93%

1.17%

500

4.67%

19.12%

4.92%

20.31%

4.00%

34.66%

5000

3.23%

44.09%

3.67%

42.00%

2.81%

55.24%

Table 2: Cytogenetic analysis of CHL cells treated with 1,1 -Dimethylurea (6 hour treatment)

Treatment

Concentration

μg/ml

S9

Cells Scored

Total number of aberrations

 

Gaps Br   Ex     Poly

Number of aberration cells

Aberration rate%

Results

Blank control

0

200

2

1

0

1

1

0.5 %

Solvent control

0.1% DMSO

 

3

1

0

2

0

0.5 %

 

50

200

2

2

0

1

2

1 %

500

200

4

3

1

1

4

2 %

5000

200

2

4

1

2

5

2.5 %

MMC

0.1

200

12

33

38

5

63

31.5 %**

Blank control

0

200

2

1

1

1

2

1 %

Solvent control

0.1% DMSO

 

4

0

1

1

1

0.5 %

 

50

200

2

1

0

1

1

0.5 %

500

200

2

2

0

1

2

1 %

5000

200

1

3

0

2

3

1.5 %

CP

10

200

15

32

22

3

55

27.5 %**

Note: **, compared with solvent control culture, P < 0.01; Br refers to chromatid break and chromosome break; Ex refers to exchange figure; MMC: Mitomycin; CP: Cyclophoshamide; Poly refers to polyploidy; +: positive; -: negative.

Table 3: Cytogenetic analysis of CHL cells treated with 1,1-dimethylurea (24hour treatment)

Treatment

Concentration

μg/ml

S9

Cells Scored

Total number of aberrations

 

Gaps Br   Ex     poly

Number of aberration cells

Aberration rate%

Results

Blank control

0

200

2

1

1

1

2

1 %

Solvent control

0.1% DMSO

200

3

1

1

2

2

1 %

 

50

200

1

2

1

1

3

1.5 %

500

200

2

4

0

5

4

2 %

5000

200

3

5

0

5

5

2.5 %

MMC

0.1

200

10

39

42

3

72

36 %**

Note: **, compared with solvent control culture, P < 0.01;Br refers to chromatid break and chromosome break;Ex refers to exchange figure; MMC: Mitomycin; CP: Cyclophoshamide;Poly refers to polyploidy; +: positive; -: negative.

Conclusions:
Under the conditions of the test 1,1-Dimethylurea was concluded to be negative for the induction of structural chromosome aberrations with and without metabolic activation using Chninese Hamster Lung cells.
Executive summary:

The substance 1,1 -Dimethylurea was tested in an in vitro Mammalian Chromosome Aberration Test using Chinese hamster lung (CHL) cells cultures. Treatments were performed both in the absence and presence of a S9 activation system. The chromosome aberration assay was used to evaluate the clastogenic potential of the test substance.

The cells were treated for 6 and 24 hours in the non-activated test system and for 6 hours in the S9-activated test system. Mammalian chromosome aberration was analyzed at three dose levels (50, 500 and 5000mg/ml), continuously for 6 hours and 24 hours. Blank control,solvent control and positive control cultures were included in the test system.

Chromosome aberrationrate of CHL in the absence of S9 and treated for 6 h was 1 %, 2 % and 2.5 % in 50, 500 and 5000 μg/ml treated groups, respectively. Chromosome aberration rate of CHL in the presence of S9 and treated for 6 h was 0.5 %,1 % and 1.5 % in 50, 500 and 5000 μg/ml treated groups,,respectively. Chromosome aberrationrate of CHL in the absence of S9 and treated for 24 h was 1.5 %, 2 % and 2.5 % in 50, 500 and 5000 μg/mL treated groups, respectively.The percentage of cells with structural aberrations in the test substance-treated groups was not increased relative to the solvent control at any dose level (p > 0.05, Chi-square test).

Based on the findings of this study,1,1 -Dimethylurea was concluded to be negative for the induction of structural chromosome aberrations in both non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHL.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Analytical purity: 100.4 %
- Impurities (identity and concentrations): < 1 % Urea, 0.1 % Isopropanol, 0.4 % diethyleneglycolmonoethylether
- Lot/batch No.: TG-OC-03-067
Target gene:
Gene involved in histidine synthesis
Species / strain / cell type:
S. typhimurium TA 97
Remarks:
a
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 102
Species / strain / cell type:
S. typhimurium TA 1535
Metabolic activation:
with and without
Metabolic activation system:
Mammalian Microsomal Fraction S9 mix
Test concentrations with justification for top dose:
Preliminary test: 5000, 1667, 556, 185, 62, 21 µg/plate
First and Second experiment: 5000, 1667, 556, 185 and 62 µg/plate
Vehicle / solvent:
The test substance was dissolved in deionised water. The solvent, deionised water, was also used for the negative control group.
Sodium azide and t-butyl-hydroperoxide were dissolved in sterile water. The other reference substances were prepared from stock solutions in dimethylsulfoxide.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
2-nitrofluorene
sodium azide
other: 2-Aminoanthracene (2AA), 1,8-Dihydroxy-anthraquinone (DHA), 4-Nitro-o-phenylenediamine (4NOPD), t-Butyl-hydroperoxide (tBHPO)
Details on test system and experimental conditions:
See chapter "any other information on materials and methods incl. tables"
Evaluation criteria:
The criteria for a positive result are: A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:
- For the strains with a low spontaneous revertant rate i.e. TA98 and TA 1535: The 2.5 fold of the amount of the spontaneous revertants.
- For the strains with a high spontaneous rate i.e. TA 97a, TA 100 and TA 102: The 1 2/3 fold of the amount of the spontaneous revertants.
These threshold values were derived from the variations in the control samples of the laboratories historic data of the Ames test.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
Key result
Species / strain:
S. typhimurium TA 97
Remarks:
a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium 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 examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
According to the results obtained in this study, 1,1 -Dimethylurea is non-mutagenic in the Ames test with the strains TA97a, TA98, TA100, TA102 and TA1535 up to 5000 µg/plate, which is the limit concentration for this kind of test.
Executive summary:

1,1 -Dimethylurea was tested for mutagenic activity with the "Salmonella typhimurium Reverse Mutation Test" (Ames Test). The study was conducted in accordance with the OECD-guideline 471 and directive 2000/32/EC, part B.13/14.

The test substance, dissolved in deionised water, was tested at concentrations ranging from 62 µg to 5000 µg per plate according to the "direct plate incorporation method" without external metabolisation as well as with an external metabolising system (S9 -mix). As test system the bacterial strains Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 were used. Negative and positive controls were included. An independent repetition of the experiment was performed.

All positive control groups showed significantly increased mutation frequencies which demonstrate the sensitivity of the test system. No toxicity of the test substance to the bacteria was observed up to 5000 µg per plate. No precipitation of the test substance was seen in any of the concentration groups. In none of the concentrations tested and with none of the strains used an increase of the mutation frequency to more than the threshold values (250 % of the controls for strains TA98 and TA1535, 167 % of the controls for strains TA97a, TA100 and TA102) was obtained. Metabolic activation did not change these results.

According to the results obtained in this study, 1,1 -Dimethylurea is non-mutagenic in the Ames test with the strains TA97a, TA98, TA100, TA102 and TA1535 up to 5000 µg/plate, which is the limit concentration for this kind of test.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11/01/2015 - 13/04/2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AlzChem AG Batch 534201
- Expiration date of the lot/batch: 31/12/2017
- Purity test date: 17/12/2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
Target gene:
Thymidine Kinase Locus/TK+/-
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Without and with metabolic activation:
0.10, 0.25, 0.5, 1.0, 2.5, 5.0, 7.5, 10 mM.
Vehicle / solvent:
A solubility test was performed with different solvents and vehicles up to the maximum recommended concentration of 10 mM.
Based on the results of the solubility test RPMI cell culture medium was used as solvent (RPMI + 5 % HS). The solution was treated with ultrasound for 10 to 15 mimnutes at 37 °C and diluted prior to treatment. The solvent was compatible with the survival of the cells and the S9 activity.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
methylmethanesulfonate
Details on test system and experimental conditions:
Cells:
Mouse Lymphoma L5178Y cells (clone TK+/- -3.7.2C) have been used successfully in in vitro experiments for many years. These cells are characterised by their high proliferation rate (10-12 h doubling time of the Eurofins Munich stock cultures) and their cloning efficiency, usually more than 50%. The cells obtain a near diploid karyotype (40 ± 2 chromosomes). They are heterozygous at the Thymidine Kinase (TK) locus in order to detect mutation events at the TK- locus.

To prevent high backgrounds arising from spontaneous mutation, cells lacking TK can be eliminated by culturing cells in RPMI 1640 supplemented with:
9.0 μg/mL hypoxanthine
15.0 μg/mL thymidine
22.5 μg/mL glycine
0.1 μg/mL methotrexate

The cells are resuspended in medium without methotrexate but thymidine, hypoxanthine and glycine for 1-3 days.
Large stock cultures of the cleansed L5178Y cell line are stored over liquid nitrogen (vapour phase)in the cell bank of Eurofins Munich. This allows the repeated use of the same cell batch inexperiments. Each cell batch is routinely checked for mycoplasma infection.
Thawed stock cultures are maintained in plastic culture flasks in RPMI 1640 complete medium and subcultured three times per week.
Rationale for test conditions:
see any other information on material and methods
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid

The test item 1,1-Dimethylurea was assessed for a possible potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The assay was conducted without and with metabolic activation. The experiment with metabolic activation was performed by including liver microsomes and NADP for efficient detection of a wide variety of carcinogens requiring metabolic activation.

The selection of the concentrations used in the main experiment was based on data from the preexperiment according to the OECD guideline 490.

In the main experiment without and with metabolic activation 10 mM was selected as the highest concentration. The experiment without and with metabolic activation was performed as a 4 h shortterm exposure assay.

The test item was investigated at the following concentrations:

without metabolic activation:

0.10. 0.25, 0.5, 1.0, 2.5, 5.0, 7.5 and 10 mM

The pH-value detected with the test item was within the physiological range.

Precipitation:

No precipitation of the test item was noted in the experiment.

Toxicity:

Slight growth inhibition was observed only in the main experiment without metabolic activation at a concentration of 0.5 mM with an RTG of 62.3. Considering that there was no evidence for a doseresponse relationship this effect was considered as not biologically relevant. In the main experiment without metabolic activation the relative total growth (RTG) was 80.8% for the highest concentration (10 mM) evaluated. The highest concentration evaluated with metabolic activation was 10 mM with a RTG of 94.8%.

Mutagenicity:

The mutant frequencies obtained from all experiments were compared with the Global Evaluation Factor (GEF) and a statistical analysis was performed. The GEF is defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data are gathered from ten laboratories. For the microwell method the GEF was defined to be 126. Criterion for mutagenicity is the extension of the GEF by the induced mutant frequency as well as a dose-dependent increase in mutant frequency. The positive controls EMS (300 μg/mL), MMS (10 μg/mL) and B[a]P (3.5 μg/mL) showed distinct effects in mutation frequency, thus proving the ability of the test system to detect potential mutagenic effects.

In the main experiment without metabolic activation all validity criteria were met. The negative controls showed mutant frequencies within the acceptance range of 50-170 mutants/106 cells, according to the IWGT criteria. The mutant frequencies of the negative control were 81.6 and 84.2 mutants/106 cells, the positive controls EMS and MMS induced a distinct increase in mutant frequency with 707.6 and 547.9 mutants/106 cells.

The mutant frequencies induced by the test item did not show a biologically relevant increase. The GEF of 126 was not exceeded in any of the dose groups showing induced mutant frequencies between -6.5 and 32.4 mutants/106 cells. None of the observed mutant frequencies was statistically significantly increased over those of the negative controls.

In the main experiment with metabolic activation all validity criteria were met. The negative controls showed mutant frequencies within the acceptance range of 50-170 mutants/106 cells, according to the IWGT criteria. The mutant frequencies of the negative control were 101.5 and 101.7 mutants/106 cells the positive control B[a]P induced a distinct increase in mutant frequency with 976.3 mutants/106 cells.

The mutant frequencies induced by the test item did not show a biologically relevant increase. The GEF of 126 was not exceeded in any of the dose groups showing induced mutant frequencies between -16.0 and 46.5 mutants/106 cells. A statistical analysis displayed that one of the mutant frequencies was significantly increased over those of the negative controls. Considering no biologically relevant increase was detected this effect was considered as not biologically relevant.

All mutant frequencies for negative and positive controls of the main experiment were found within the historical range of the test facility Eurofins Munich.

Clastogenicity:

Colony sizing was performed for the highest concentrations of the test item and for the negative and positive controls. An extension of the GEF by the induced mutant frequency in combination with an increased occurrence of small colonies (defined by slow growth and/or morphological alteration of the cell clone) is an indication for potential clastogenic effects and/or chromosomal aberrations. The positive controls MMS (10 μg/mL) and B[a]P (3.5 μg/mL) induced a significant increase in mutant frequency and a biologically significant increase of small colonies (≥40%), thus proving the ability of the test system to indicate potential clastogenic effects.

In the main experiment without metabolic activation the percentage of small colonies in the negative controls was found to be 6.3% and 3.0%. The percentage of small colonies in the positive control MMS was found to be 42.7%. In the highest dose groups 16.9% (5.0 mM), 16.1% (7.5 mM) and 10.9% (10 mM) of small colonies were found. As none of the values exceeded 40%, all dose groups were considered as not clastogenic.

With metabolic activation the percentage of small colonies in the negative controls was found to be 16.4% and 9.1%. The percentage of small colonies of the positive control B[a]P was found to be 40.4%. In the highest dose groups 9.0% (5.0 mM), 10.0% (7.5 mM) and 12.0% (10 mM) of small colonies were found. As none of the values exceeded 40%, all dose groups were considered as not clastogenic.

Conclusions:
In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item 1,1-Dimethylurea is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.
Executive summary:

The test item 1,1 -Dimethylurea was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y according to OECD 490.

The selection of the concentrations used in the main experiment was based on data from the pre-experiment. In the main experiment without and with metabolic activation 10 mM was selected as the highest concentration. The experiment without and wit metabolic activation was performed as a 4 h short-term exposure assay.

The test item was investigated at the following concentrations:

without and with metabolic activation:

0.10, 0.25, 0.5, 1.0, 2.5, 5.0, 7.5, and 10 mM

No precipitation of the test item was noted in the experiment.

No biologically relevant growth inhibition was observed in the experiment.

In the main experiment without metabolic activation the relative total growth (RTG) was 80.8 % for the highest concentration (10 mM) evaluated. The highest concentration evaluated with metabolic activation was 10 mM with a RTG of 94.8 %.

In the main experiment no biologically relevant increase of mutants was found after treatment with the test item (without and with metabolic activation). The Global Evaluation Factor (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories) was not exceeded by the induced mutant frequency at any concentration.

No dose-response relationship was observed.

Additionally, in the main experiment colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (without and with metabolic activation).

Ethylmethynesulfonate, Methylmethanesulfonate and Benzo[a]pyrene were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Aditionally,

Ethylmethynesulfonate, Methylmethanesulfonate and Benzo[a]pyrene significantly increased the number of small colonies, thus providing the efficiency of the test system to indicate potential clastogenic effects.

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item 1,1-Dimethylurea is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

In accordance with the above named results of several tests on the genetic toxicity 1,1 -Dimethylurea does not have to be classified.