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Toxicological information

Genetic toxicity: in vitro

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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-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
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 / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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
Controlsopen allclose all
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
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9

Migrated to IUCLID6: 80 µg/plate for TA1537
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
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
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 resultsopen allclose all
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
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.