Methylurea

Genetic toxicity: in vitro

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

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

Test material information

Constituent 1

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 .

Controls

Controls 1

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

Controls 2

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

Controls 3

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

Controls 4

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