Hydrocarbons, C4, 1,3-butadiene-free, polymd., tetraisobutylene fraction

Genetic toxicity: in vitro

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

Study period:

22 January - 10 February 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, guideline study available as unpublished report. Fully adequate for assesment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: EPA (TSCA) OPPTS harmonised guidelines

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Butene, homopolymer (products derived from either/or But-1-ene/But-2-ene)

EC Number:

500-004-7

EC Name:

Butene, homopolymer (products derived from either/or But-1-ene/But-2-ene)

Cas Number:

9003-29-6

Molecular formula:

C8H16

IUPAC Name:

Butene, homopolymer (products derived from either/or But-1-ene/But-2-ene)

Constituent 2

Reference substance name:

tetrabutene

IUPAC Name:

tetrabutene

Details on test material:

- Name of test material (as cited in study report): tetrabutene
- Physical state: Clear colourless liquid
- Storage condition of test material: Room temperature in the dark under nitrogen
- Other: The integrity of supplied data relating to the identity, purity and stability of the test material is the responsibility of the Sponsor.

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:

Liver S9 from phenobarbitone/β-naphthoflavone induced rats

Test concentrations with justification for top dose:

50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle: tetrahydrofuran
- Justification for choice of solvent/vehicle: The test material was immiscible in sterile distilled water, dimethyl sulphoxide, acetone, dimethyl formamide and acetonitrile at 50 mg/mL but was fully miscible in tetrahydrofuran at 200 mg/mL in solubility checks performed in-house.

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9

Migrated to IUCLID6: 3 µg/plate for TA100, 5 µg/plate for TA1535 and 2 µg/plate for WP2uvrA

Controls 2

Positive control substance:

9-aminoacridine

Remarks:

without S9

Migrated to IUCLID6: 80 µg/plate for TA1537

Controls 3

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9

Migrated to IUCLID6: 0.2 µg/plate for TA98

Controls 4

Positive control substance:

other: 2-aminoanthracene: 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537, and 10 µg/plate for WP2uvrA

Remarks:

with S9

Controls 5

Positive control substance:

benzo(a)pyrene

Remarks:

with S9

Migrated to IUCLID6: 5 µg/plate for TA98

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Five concentrations of the test material (50, 150, 500, 1500 and 5000 µg/plate) were assayed using the direct plate incorporation method.
- Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.025 mL of the vehicle or test material formulation or 0.1 mL of positive control and either 0.5 mL of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate).

DURATION:
- Preincubation period: approximately 48 hrs at 37°C

NUMBER OF REPLICATIONS: This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.

Other:
- A second experiment was performed using methodology as described for Experiment 1, using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as Experiment 1 (50 to 5000 µg/plate).

Evaluation criteria:

Several criteria applied for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation.

Results and discussion

Test results

Test results 1

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:

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

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

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
- In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test material.
- The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
- The test was performed by mixing 0.1 mL of bacterial culture (TA100 or WP2uvrA), 2 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.025 mL of test material formulation and 0.5 mL of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). Ten concentrations of the test material formulation and a vehicle control (tetrahydrofuran) were tested. In addition, 0.025 mL of the maximum concentration of the test material and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material.
- After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.
- The test material was non-toxic.

ACCEPTANCE CRITERIA (main study):
- The following acceptance criteria were met: The appropriate characteristics for each tester strain were confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
- All tester strain cultures were in the range of 1 to 9.9 x 109 bacteria per mL.
- Each mean positive control value was at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
- There was a minimum of four non-toxic test material dose levels.
- There was no evidence of excessive contamination.

Any other information on results incl. tables

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A fine, particulate precipitate was observed at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Tetrabutene was considered to be non-mutagenic under the conditions of this test.

Executive summary:

In an Ames assay with S. Typhimurium TA1535, TA1537, TA98, TA100 and E.coli WP2uvrA, tetrabutene (CAS No. 9003-29-6) was considered to be non-mutagenic under the conditions of the test.