1,1-dimethylurea

Genetic toxicity: in vitro

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 guideline

Test guideline 1

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)

Test guideline 2

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)

Test guideline 3

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

Test material information

Constituent 1

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

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

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)	-S9（6h） Mitotic index	- S9（6h） inhibition rate	+ S9（6h） Mitotic index	+ S9（6h） inhibition rate	- S9（24h） Mitotic index	- S9（24h） 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.