Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Chemical structure
Reference substance name:
1,1-dimethylurea
EC Number:
209-957-0
EC Name:
1,1-dimethylurea
Cas Number:
598-94-7
Molecular formula:
C3H8N2O
IUPAC Name:
1,1-dimethylurea
impurity 1
Chemical structure
Reference substance name:
Urea
EC Number:
200-315-5
EC Name:
Urea
Cas Number:
57-13-6
Molecular formula:
CH4N2O
IUPAC Name:
urea
impurity 2
Chemical structure
Reference substance name:
Water
EC Number:
231-791-2
EC Name:
Water
Cas Number:
7732-18-5
Molecular formula:
H2O
IUPAC Name:
dihydrogen oxide
Test material form:
solid: crystalline
Specific details on test material used for the study:
- Analytical purity: 99.9 %
- Lot/batch No.: 301601

Method

Species / strain
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
Controls
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.

Results and discussion

Test results
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"

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.

Applicant's summary and conclusion

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.