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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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
N,N''-hexane-1,6-diylbis[N'-benzylurea]
EC Number:
254-274-3
EC Name:
N,N''-hexane-1,6-diylbis[N'-benzylurea]
Cas Number:
39072-70-3
Molecular formula:
C22H30N4O2
IUPAC Name:
3-benzyl-1-{6-[(benzylcarbamoyl)amino]hexyl}urea
Details on test material:
- Name of test material (as cited in study report): N,N''-Hexane-1,6-diylbis[N'-benzylurea]
- Physical state: solid
- Purity: 1H-NMR spectroscopy: 98.0 g/100 g, HPLC fingerprint: 90.1 area-%
- Test-substance No.: 11/0726-1
- Lot/batch No.: 259-635
- Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions.
- Storage stability: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility

Method

Target gene:
HIS/TRP
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
33 μg - 5 000 μg/plate (SPT)
33 μg - 5 000 μg/plate (PIT)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: [DMSO]
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in ultrapure water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: With S9 mix: 2-aminoanthracene (2-AA); Without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylenediamine (NOPD), 9-aminoacridine (AAC), 4-nitroquinoline-N-oxide (4-NQO)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Incubation period: 20 min in medium (PIT) and incubation at 37°C for 48 – 72 hours (PIT and SPT)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
The test substance is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.
Statistics:
not applicable

Results and discussion

Test resultsopen allclose all
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:
cytotoxicity
Remarks:
A bacteriotoxic effect was observed depending on the strain and test conditions from about 2 500 μg/plate onward
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A bacteriotoxic effect was observed depending on the strain and test conditions from about 2 500 μg/plate onward
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Bacteriotoxicity (decrease in the number of his+ or trp+ revertants, slight reduction in the titer) was observed in the standard plate test and in the preincubation assay depending on the strain and test conditions from about 2 500 μg/plate onward.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

According to the results of the present study, the test substance did not lead to a relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard

plate test and preincubation assay).

Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study.

In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.

In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above.

Applicant's summary and conclusion