Reaction mass of 2-(3-{6-[3-(2-Hydroxyethyl)-3-methylureido]hexyl}-1-methylureido)ethanol and 6-[3-(2-Hydroxyethyl)-3-methylureido]hexylamino 3-(methylamino)propionate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N''-hexane-1,6-diylbis[N'-(2-hydroxyethyl)-N'-methylurea]

EC Number:

253-281-9

EC Name:

N,N''-hexane-1,6-diylbis[N'-(2-hydroxyethyl)-N'-methylurea]

Cas Number:

36938-15-5

Molecular formula:

C14H30N4O4

IUPAC Name:

2-(3-{6-[3-(2-Hydroxyethyl)-3-methylureido]hexyl}-1-methylureido)ethanol

Constituent 2

Reference substance name:

6-[3-(2-Hydroxyethyl)-3-methylureido]hexylamino 3-(methylamino)propionate

Molecular formula:

C14H30N4O4

IUPAC Name:

6-[3-(2-Hydroxyethyl)-3-methylureido]hexylamino 3-(methylamino)propionate

Test material form:

solid: bulk

Details on test material:

- Batch no.: EGGE 2806-1
- Currenta sample no.: 2187

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Stable in deionised water for at least 4 hours at 20 ± 5°C (light-protected) ((Envigo Study Number D98915, 26st of February 2015)


FORM AS APPLIED IN THE TEST (if different from that of starting material): On the day of the experiment, the test item was suspended in deionised water. The formulation was prepared freshly before treatment and used within two hours of preparation.

Method

Target gene:

mutant histidine gene

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

plate incorporation assay:
3, 10, 33, 100, 333, 1000, 2500, 5000 µg/plate with and without S9 mix
preincubation assay:
3, 10, 33, 100, 333, 1000, 2500, 5000 µg/plate with and without S9 mix

The test was performed up to and including the limit dose of 5000 µg/plate and tube. No precipitation occurred up to and including 5000 µg/plate. The plates incubated with the test item showed normal background growth up to 5000 μg/plate in all strains used. Only in experiment I in strains TA 1537, TA 98 and TA 100 with S9 mix reduced background growth was observed at 5000 μg/plate.
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation up to and including 5000 μg/plate.

Vehicle / solvent:

Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used.

DETERMINATION OF TOXICITY
- Method: Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Only in the plate incorporation test with S9 mix reduced background growth was observed at the limit concentration - no toxic effects, evident as a reduction in the number of revertants occurred.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Only in the plate incorporation test with S9 mix reduced background growth was observed at the limit concentration - no toxic effects, evident as a reduction in the number of revertants occurred.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Only in the plate incorporation test with S9 mix reduced background growth was observed at the limit concentration - no toxic effects, evident as a reduction in the number of revertants occurred.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

negative

Executive summary:

The test item was evaluated in an Ames Test on Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA, performed according to OECD TG 471. No precipitation of the test item occurred up to and including the highest investigated dose (5000 µg/plate). The plates incubated with the test item showed normal background growth up to 5000 μg/plate in all strains used. Only in experiment I in strains TA 1537, TA 98 and TA 100 with S9 mix reduced background growth was observed at 5000 μg/plate. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation up to and including the highest investigated dose. The test material was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test. Positive controls increased the mutant counts to well over those of the negative controls, thus demonstrated the system´s sensitivity.