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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP guideline study. However, only 4 strains of Salmonella typhimurium were used; testing with Salmonella typhimurium TA102 or Escherichia coli was not carried out. Therefore, the study does not completely fulfill the reliability criteria of today's guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guidelineopen allclose all
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
Remarks:
with the deviation, that analytical investigations of the stability of the test substance in aqua dest. were not carried out.
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Methylurea
EC Number:
209-935-0
EC Name:
Methylurea
Cas Number:
598-50-5
Molecular formula:
C2H6N2O
IUPAC Name:
methylurea
Details on test material:
- Name of test material (as cited in study report): Methylharnstoff, technisch (methylurea, technical grade),
- Number of the test substance: 91/85
- Physical state / colour: crystalline, white
- Analytical purity: 98.5%
- Lot/batch No.: 08710
- Stability under test conditions: Reanalysis at a later date, results not given in this report.
- Storage condition of test material: Room temperature (tightly closed and protected from light)

Method

Target gene:
the amino acid histidine locus
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix from liver homogenate of 5 Arochlor 1254 pretreated male Sprague-Dawley rats
Test concentrations with justification for top dose:
1st experiment: TA 100 and TA 98: 0, 20, 100, 500, 2500 and 5000 µg/plate.
2nd experiment:TA 98: 0, 500, 1000, 2000, 3000 and 4000 µg/plate.
3rd experiment: TA 1535 and TA 1537: 0, 20, 100, 500, 2500 and 5000 µg/plate.
4th experiment: TA 1535, TA 1537, TA 98 and TA 100: 0, 20, 100, 500, 2500 and 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Complete solubility of the test substance in aqua dest .
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
10 µg (dissolved in DMSO) was used in the presence of S-9 mix in all investigated strains
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)
Remarks:
5 µg (dissolved in DMSO) was used without the presence of S-9 mix in the strains TA 100 and TA 1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylendiamine
Remarks:
10 µg (dissolved in DMSO) was used without the presence of S-9 mix in strain TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 9-aminoacridine chloride monohydrate
Remarks:
100 µg (dissolved in DMSO) was used without the presence of S-9 mix in strain TA 1537
Details on test system and experimental conditions:
METHOD OF APPLICATION:
• Standard plate test (SPT): in agar (plate incorporation), [ Experiment 1 - 3] and
• preincubation test (PIT), [ Experiment 4]
Both with and without metabolic activation with S-9 mix prepared from liver homogenate of Aroclor 1254-pretreated male Sprague Dawley rats.

DURATION
- Preincubation period:
• In the plate incorporation plate test (standard plate test) there is no preincubation period (with the test substance).
• In the preincubation test: 20 min., before the mixture of the bacteria, test substance and medium are poured to the selective minimal agar.
- Exposure duration: 48 hours
- Selection time (if incubation with a selection agent): 48 hours

SELECTION AGENT (mutation assays): Minimal agar without histidine

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (reduced background growth of histidine-negative colonies)

Evaluation criteria:
In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- Doubling of the spontaneous mutation rate (control)
- Dose-response relationship
- Reproducibility of the results
Statistics:
The mean number of revertant colonies per plate, titer, and the standard deviations were calculated for all dose groups as well as for the positive and negative (vehicle) controls in all experiments.


Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Standard plate test (20 - 5000 µg/plate)

Strain

Metabolic activation system

mean revertants in Controls

maximum revertant factor

dose dependency

Assessment

TA 98

1st Exp

no

29

1.0

no

negative

yes

39

1.4

no

negative

TA 98

2nd Exp

no

24

1.4

no

negative

yes

32

1.3

no

negative

TA 1537 

no

13

0.8

no

negative

yes

15

1.1

no

negative

TA 1535 

no

21

1.0

no

negative

yes

24

0.9

no

negative

TA 100 

no

108

1.1

no

negative

yes

115

1.3

no

negative

Preincubation test (20 - 5000 µg/plate)

Strain

Metabolic activation system

mean revertants in Controls

maximum revertant factor

dose dependency

Assessment

TA 98

no

16

1.4

no

negative

yes

23

1.2

no

negative

TA 1537

no

13

0.9

no

negative

yes

14

1.2

no

negative

TA 1535

no

18

0.9

no

negative

yes

15

1.1

no

negative

TA 100

no

95

1.2

no

negative

yes

114

1.1

no

negative

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative