Oxazolidine, diheptyl~

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:

Dec. 2006 - Jan. 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and ß-naphthoflavone induced rat liver S9-mix

Test concentrations with justification for top dose:

DOSE RANGE FINDING TEST: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
MUTATION ASSAY: 10, 33, 100, 333 and 1000 µg/plate

Precipitation of SAT 060283 on the plates was observed at the start and at the end of the incubation period at concentrations of 1000 µg/plate and upwards.

Vehicle / solvent:

The test substance was dissolved in dimethyl formamide (Merck, Darmstadt, Germany). Test substance concentrations were used within 3 hours after preparation.

Controls

Controls 1

Untreated negative controls:

other: solvent control served as negative control

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 2-nitrofluorene

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

other: solvent control served as negative control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

The characteristics of the different Salmonella typhimurium strains were as folIows:
- TA1537: hisC3076 Frameshift
- TA 98: hisD3052 / R-factor*, Frameshift
- TA 1535: hisG46, Base-pair substitutions
- TA 100: hisG46 / R-factor*, Base-pair substitutions
(*R-factor = plasmid pKM101 (increases error-prone DNA repair))

Each tester strain contained the following additional mutations:
rfa : deep rough (defective Iipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)

The Salmonella typhimurium strains were regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.

The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using Tris-EDTA treatment. The strain was regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants.

Stock cultures of the five strains were stored in liquid nitrogen (-196°C).

Evaluation criteria:

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain.
b) The positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive contral substance. Furthermore, the mean plate count should be at least three times the concurrent vehicle control group mean.
c) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA 100 is not greater than two times the concurrent control, and the total number of revertants in tester strains TA 1535, TA 1537, TA98 or WP2uvrA is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA 100 is greater than two times the concurrent control, or the total number of revertants in tester strains TA 1535, TA1537, TA98 or WP2uvrA is greater than three times the concurrent control.
b) In case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

No formal hypothesis testing was done.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, T A98, TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

Dose range finding test

The test substance was tested in the tester strains TA 100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix.

- Precipitate: Precipitation of the test substance on the plates was observed at the start and at the end of the incubation period at concentrations of 1000 µg/plate and upwards.

- Toxicity: To determine the toxicity of the test substance, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. No reduction of the bacterial background lawn was observed at any of the concentrations tested in both tester strains.

In strain TA 100 in the presence of S9-mix, fluctuations in the number of revertant colonies below the laboratory background historical control data range were observed. However, since no dose-relationship was observed, the reductions are not considered to be caused by toxicity of the test substance. In strain WP2uvrA, no biologically significant decrease in the number of revertants was observed.

- Mutagenicity: In the dose range finding test, no increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.

Mutation assay

Based on the results of the dose range finding test, the test substance was tested up to concentrations of 1000 µg/plate in the absence and presence of 89-mix in two mutation assays.

The first mutation experiment was performed with the strains TA 1535, TA 1537 and TA 98 and the second mutation experiment was performed with the strains TA 1535, TA 1537, TA 98, TA 100 and WP2uvrA.

- Precipitate: Precipitation of the test substance on the plates was observed at the start and at the end of the incubation period at the concentration of 1000 µg/plate.

- Toxicity: In both mutation assays, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.

- Mutagenicity: In both mutation assays, no increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that the est substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia eali reverse mutation assay.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Evaluation of the mutagenic activity of the test substance in the S. typhimurium reverse mutation assay and the E. coli reverse mutation assay (with independent repeat)

The test substance was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone) .

The study procedures described in this report were based on the most recent OECD and EEC guidelines.

The tesat substance was a clear yellowish liquid with a purity of 90%. The test substance was dissolved in dimethyl formamide.

In the dose range finding test, the test substance was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA 100 and WP2uvrA. The test substance precipitated on the plates at dose levels of 1000 µg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

Based on the results of the dose range finding test, the test substancewas tested in the first mutation assay at a concentration range of 10 to 1000 µg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA 1535, TA 1537 and TA98. In an independent repeat of the assay with additional parameters, the test substance was tested at the same concentration range as the first assay in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. SAT 060283 precipitated on the plates at the top dose of

1000 µg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

The test substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the

number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated

experiment.

In this study, the negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the

metabolic activation system functioned properly.

Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation

assay.