Oligomerisation products of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane with acrylic acid and fatty acids, C18-unsatd., dimers and nonanoic acid

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:

From November 20, 2011 to December 14, 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2012

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:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

Oligomerisation products of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane with acrylic acid and fatty acids, C18-unsatd., dimers and nonanoic acid

EC Number:

701-359-2

Cas Number:

216689-76-8

Molecular formula:

Molecular formula not available for this UVCB.

IUPAC Name:

Oligomerisation products of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane with acrylic acid and fatty acids, C18-unsatd., dimers and nonanoic acid

Test material form:

liquid

Specific details on test material used for the study:

The test substance was dissolved in dimethyl sulfoxide (SeccoSolv, Merck, Darmstadt, Germany). The stock solution was treated with ultrasonic waves until the test substance had completely dissolved. Test substance concentrations were used within approximately 1½ hours after preparation.

Method

Target gene:

- Histidine
- Tryptophan

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

The strains TA1537 and TA98 are capable of detecting frameshift mutagens, strains TA1535, TA100 and WP2uvrA are capable of detecting base-pair substitution mutagens.

Metabolic activation:

with and without

Metabolic activation system:

metabolizing system (S9-mix)

Test concentrations with justification for top dose:

Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. Eight concentrations, 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate were tested in triplicate. This dose range finding test was reported as a part of the first experiment of the mutation assay. The highest concentration of the test substance used in the subsequent mutation assay was the level at which the test substance exhibited limited solubility.

Vehicle / solvent:

dimethyl sulfoxide

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle alone: dimethyl sulfoxide

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191, 2-aminoantracene

Details on test system and experimental conditions:

Salmonella typhimurium bacteria and Escherichia coli bacteria.
Source:
- Salmonella typhimurium strains: Trinova Biochem GmbH, Germany (Master culture from Dr. Bruce N. Ames): TA100, TA98, TA1537, TA1535
- Escherichia coli strain: Trinova Biochem GmbH, Germany (Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK) WP2uvrA
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).

Rationale for test conditions:

Recommended test system in international guidelines (e.g. OECD and EC).

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 control 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.

Statistics:

No formal hypothesis testing was done.
- A test substance is considered negative (not mutagenic) in the test if: a) The total number of revertants in tester strain TA100 is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three times the concurrent vehicle 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 TA100 is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three times the concurrent vehicle control. b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, 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.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Experiment 1:
Based on the results of the dose range finding test, the test substance was tested up to the dose level of 3330 µg/plate in the absence and presence of 5% (v/v) S9-mix with the Salmonella typhimurium strains, TA1535, TA1537 and TA98. Precipitation of the test substance on the plates was observed at the start and at the end of the incubation period at the highest dose level of 3330 µg/plate. With the exception of tester strain TA98 in the absence of S9-mix (where a moderate reduction in the number of revertant colonies was observed at the two highest tested concentrations), 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 three tester strains in the absence and presence of S9-mix. Finally, no increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.
- Experiment 2:
To obtain more information about the possible mutagenicity of the test substance, a second mutation experiment was performed in the absence of S9-mix and in the presence of 10% (v/v) S9-mix. Based on the results of the first mutation assay, the test substance was tested up to the dose level of 3330 µg/plate in strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Precipitation of the test substance on the plates was observed at the start of the incubation period at a concentration of 3330 µg/plate. At the end of the incubation period precipitation was observed at 1000 and 3330 µg/plate in all tester strains, with the exception of tester strain WP2uvrA. In this tester strain precipitation on the plates was already observed at a dose level of 333 µg/plate. With the exception of tester strain TA100 in the presence of S9-mix (where an extreme reduction in the number of revertant colonies was observed at the highest tested concentration), 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. Finally, in the second mutation experiment, no biological relevant increase in the number of revertants was observed upon treatment with the test substance under all conditions tested. Although an increase above the historical control data range was observed at a dose of 333 µg/plate in tester strain TA1537 in the presence of S9-mix, this increase was not dose dependent and caused by one outlier. Therefore this increase was considered not biologically relevant.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, it was concluded that the test substance was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

A study was conducted to determine the mutagenic potential of the test substance according OECD Guideline 471 and EU Method B.13/14. The substance was tested up to the dose level of 3330 µg/plate in strains TA1535, TA1537, TA98, TA100 and WP2uvrA with (5 or 10% (v/v)) and without metabolic activation (S9-mix). Precipitates, toxicity and mutagenicity were evaluated. All bacterial strains showed negative responses over the entire dose range, i.e. there was 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. Under the study conditions, the substance was not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assays (Verbaan, 2011).