1,6-hexanediyl-bis(2-(2-(1-ethylpentyl)-3-oxazolidinyl)ethyl)carbamate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990-09-18 to 1990-09-27

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP and guideline study. No data were available on analytical inveatigations on the stability of the test substance in solution. However, this deviation did not limit the assessment of the results. Only four strains tested.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Directive 84/449/EEC

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 26 May 1983

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,6-hexanediyl-bis(2-(2-(1-ethylpentyl)-3-oxazolidinyl)ethyl)carbamate

EC Number:

411-700-4

EC Name:

1,6-hexanediyl-bis(2-(2-(1-ethylpentyl)-3-oxazolidinyl)ethyl)carbamate

Cas Number:

140921-24-0

Molecular formula:

C32H62N4O6

IUPAC Name:

2-[3-(heptan-3-yl)-1,2-oxazolidin-2-yl]ethyl N-{6-[({2-[3-(heptan-3-yl)-1,2-oxazolidin-2-yl]ethoxy}carbonyl)amino]hexyl}carbamate

Test material form:

liquid

Method

Target gene:

Histidine-auxotrophic Salmonella typhimurium strains TA 1535, TA 100, TA 1537 and TA 98.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 fraction

Test concentrations with justification for top dose:

With S9 mix: 8, 40, 200, 1000, 5000 µg/plate
Without S9 mix: 8, 40, 200, 1000, 5000 µg/plate

Vehicle / solvent:

The solvent employed for the test substance was ethanol and for the positive controls DMSO.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

in strain TA1535, without S9 mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: nitrofurantoin

Remarks:

in strain TA100, without S9 mix

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene diamine

Remarks:

in strains TA1537 and TA 98, without S9 mix

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

all strains, with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours at 37 °C

SELECTION AGENT: histidine

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

The following criteria were used for the acceptance of an assay:
a) The negative controls have to be within the expected range, as defined by literature data and the laboratories' own historical data.
b) The positive controls have shown sufficient effects as defined by the laboratories' experience.
c) The titter determinations had to demonstrate sufficient bacterial density in the suspension.
An assay which was not in agreement with at least one of the above criteria was not used for assessment.
Furthermore the data generated in this assay have to be confirmed by two additional independent experiments. Even, if the criteria for points a, b and c were not met, an assay was accepted if it showed mutagenic activity of the test substance.

Evaluation criteria:

A reproducible and dose-related increase in mutant counts for at least one strain is considered to be a positive result.
For TA 1535, TA 100 and TA 98 this increase should be about twice the amount of negative controls, whereas for TA 1537, at least a threefold increase should be reached. Otherwise the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgement. In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Statistics:

not applicable

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2

Metabolic activation:

not specified

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

not examined

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

There was no indication of a bacteriotoxic effect for the test substance at doses up to and including 200 µg per plate. The total bacteria counts consistently produced results comparable to the negative control, or differed only insignificantly. Likewise an inhibition of growth was not detected.
Higher doses had a strong, strain-specific bacteriotoxic effect. Therefore they could only be used to a limited extent up to 5000 µg per plate.
None of the four strains concerned showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to the test with and without S9 mix and was confirmed by the results of the repeated tests.

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions, the test substance showed no mutagenic activity in this bacterial reverse mutation test on Salmonella typhimurium.

Executive summary:

The test substance was investigated in the Salmonella / microsome test for point-mutagenic effects in doses up to 5000 µg per plate on four Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537 and TA 98. Doses up to and including 200 µg per plate did not cause any bateriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed.

At higher doses, the substance had a strain-specific bacteriotoxic effect, so that this range could only be used to a limited extend up to 5000 mg per plate for assessment purposes. Evidence of mutagenic activity for the test substance was not seen. No biologically-relevant increase of the mutant count, in comparison with the negative controls, was observed. Therefore, the test substance was considered to be non-mutagenic without and with S9 mix in the Salmonella/microsome test. The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2 -phenylene-diamine and 2 -aminoanthracene had a marked mutagenic effect, as was seen by a biologically-relevant increase in mutant colonies compared to the corresponding negative controls.