Buta-1,2-diene

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as published report, no restrictions, fully adequate for assessment

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Buta-1,2-diene

EC Number:

209-674-2

EC Name:

Buta-1,2-diene

Cas Number:

590-19-2

Molecular formula:

C4H6

IUPAC Name:

buta-1,2-diene

Constituent 2

Reference substance name:

1,2 butadiene

IUPAC Name:

1,2 butadiene

Details on test material:

- Name of test material (as cited in study report): 1,2 butadiene
- Physical state: clear colourless gas
- Storage condition of test material: room temperature in the dark
- no further data

Method

Target gene:

not applicable

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:

phenobarbitone/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

1.5, 3.0, 6.0, 9.0 and 12% (air mixtures)

Vehicle / solvent:

clean, dry air

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

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9

Migrated to IUCLID6: 80 µg/plate for TA1537

Controls 3

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9

Migrated to IUCLID6: 0.2 µg/plate for TA98

Controls 4

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene; at 1 µg/plate for TA100 and 2 µg/plate for TA1535 and TA1537. At 10 µg/plate for WP2uvrA

Remarks:

with S9

Controls 5

Negative solvent / vehicle controls:

yes

Positive controls:

yes

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 (1.5, 3.0, 6.0, 9.0, and 12.0%) were assayed in triplicate against each tester strain
- 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 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).
- The plates were then allowed to set and placed into gas canisters with inlet and outlet valves.
- Atmospheres of the test material were generated and passed into the gas canisters containing the agar plates (containing the tester strains and S9-mix or phosphate buffer).
- The gas canisters were then placed in an incubator at 37°C.
- 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.

NUMBER OF REPLICATIONS:
- The procedure was repeated, in triplicate, for each bacterial strain both with and without S9-mix.

TOXICITY TEST:
- In order to select appropriate dose levels for use in the main test, a preliminary test was carried in strains TA100 and WP2uvrA out to determine the toxicity of the test material. The concentrations tested were 1.5, 3.0, 6.0, 12.5 and 50.0%.

OTHER:
- Prior to the master strains being used, characterisation checks were carried out to confirm the amino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate.

Evaluation criteria:

There are several criteria 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.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

None specified.

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:

other: no, but tested up to Upper Explosive Limit of 12% (265,000mg/m3)

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:

other: no, but tested up to Upper Explosive Limit (265,000mg/m3)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA).

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. The test material was, therefore, tested up to the maximum recommended dose level of 12.0% (Upper Explosive Limit).

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

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

1,2-Butadiene was non-mutagenic in a bacterial mutation assay.

Executive summary:

1,2-Butadiene was tested in a bacterial mutation assay (Ames test) up to the upper explosive limit (12% or 265,521 mg/m3). 1,2-Butadiene was non-mutagenic when tested in the presence and absence of metabolic activation.