Hydrocarbons, C4, 1,3-butadiene-free, polymd., dibutylene fraction, hydrogenated

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, guideline, fully adequate for assessment

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:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Isooctene

EC Number:

234-294-9

EC Name:

Isooctene

Cas Number:

11071-47-9

IUPAC Name:

6-methylhept-1-ene

Details on test material:

- Name of test material (as cited in study report): isooctene
- Physical state: clear liquid
- Analytical purity: 99.80%
- Lot/batch No.: 16.06.2008
- Expiration date of the lot/batch: July 2009
- Storage condition of test material: room temperature
- Sponsor's ID number: 0649/82533

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: histidine-dependent auxotrophic mutants

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Additional strain / cell type characteristics:

other: tryptophan-dependent mutant

Metabolic activation:

with and without

Metabolic activation system:

S9 from phenobarbital and 5,6-benzoflavone induced rat liver

Test concentrations with justification for top dose:

Limit test - up to 5000 µg/plate (and a series of ca half-log10 dilutions)

Vehicle / solvent:

Dimethyl formamide (DMF)

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: sodium azide, 9-aminoacridine, 2-nitrofluorene and 4-nitroquinoline-N-oxide

Remarks:

without S9

Controls 2

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMF

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene and benzo(a)pyrene

Remarks:

with S9

Details on test system and experimental conditions:

Test 1: Aliquots of 0.1 mL of the test substance solutions (seven concentrations up to 5000 µg/plate), positive control or negative control were placed in glass vessels. The negative control was the chosen vehicle, DMF. Untreated controls were also prepared. S9 mix (0.5 mL) or 0.1 M pH 7.4 phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10-hour bacterial culture and 2 mL of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar. Each Petri dish was individually labelled with a unique code, identifying the contents of the dish. Three Petri dishes were used for each treatment. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate buffer. All plates were incubated at approximately 37°C for ca 72 hours. After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer).
Test 2: As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The pre-incubation procedure is not suitable for DMF, which is toxic under such conditions. The variation used was, therefore, an increase in the S9 content of the S9 mix from 10% v/v to 20% v/v. In the absence of S9 mix, the test procedure was the same as in test 1. The maximum concentration chosen was again 5000 µg/plate, but only five concentrations were used.

Evaluation criteria:

If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls, with some evidence of a positive dose-response relationship, it is considered to exhibit mutagenic activity in this test system. If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system.

Statistics:

No statistical analysis performed.

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A pKM 101

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

No evidence of mutagenic activity was seen at any concentration of Isooctene in either mutation test.

The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

Applicant's summary and conclusion

Conclusions:

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

Isooctene showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Executive summary:

In an in vitro assessment of the mutagenic potential of Isooctene, histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), were exposed to Isooctene diluted in dimethyl formamide (DMF). DMF was used as a negative control. Untreated controls were also included.

Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone.  Both tests were standard plate incorporation assays.

Concentrations of Isooctene up to 5000 µg/plate were tested. Other concentrations used were a series of ca half-log₁₀dilutions of the highest concentration. No signs of toxicity were observed towards the tester strains in either mutation test.

No evidence of mutagenic activity was seen at any concentration of Isooctene in either mutation test.

The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. The mean revertant colony counts for the vehicle and untreated controls were within or close to the 99% confidence limits of the current historical control range of the laboratory.

It is concluded that Isooctene showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.