2,6-diisopropylnaphthalene

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:

25 Sep - 04 Nov 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,6-diisopropylnaphthalene

EC Number:

246-045-1

EC Name:

2,6-diisopropylnaphthalene

Cas Number:

24157-81-1

Molecular formula:

C16H20

IUPAC Name:

2,6-diisopropylnaphthalene

Method

Target gene:

his operon for S. typhimurium strains and trp operon for E. coli strain

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:

Type and composition of metabolic activation system: Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Sprague-Dawley rats treated with 500 mg/kg bw Aroclor 1254.
- source of S9 : Molecular Toxicology, Inc., USA, Batch 0745 (33.15 mg of protein per mL) and Batch 0781 (40.7 mg of protein per mL)
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): yes

Test concentrations with justification for top dose:

First and second experiment: 100, 250, 500, 1000, 2500, 5000 µg/plate, with and without metabolic activation
Dose levels selected based on a dose range finding study

Vehicle / solvent:

- Vehicle: Dimethylformamide (DMF)
- Source: Sigma Chemical Co., USA (Lot 125H3633, Lot 37H3649, and Lot 47H3674)
- Justification for choice of solvent/vehicle: The solvent was chosen due to the limited solubility of the test substance

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

other: 2-aminoanthracene, 2.5 µg/plate for S. typhimurium strains, 25 µg/plate for E. coli strain, +S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments . 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)
- Exposure duration and conditions: 48 ± 8 h at 37 ± 2 °C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Inspection of the bacterial background lawn

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Counting of revertant colonies

Evaluation criteria:

The test material may be considered positive in this test system if the following criteria are met: For the test item to be considered mutagenic, two-fold (three-fold for TA 1535 and TA 1537) increases in mean revertant numbers must be observed. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels. All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.

Statistics:

Mean values and standard deviation were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitation at and above 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitation at and above 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitation at and above 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitation at and above 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitation at and above 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test substance is poorly soluble in aqueous solutions. Therefore, DMF was used as vehicle..
- Precipitation and time of the determination: At a concentration of 1000 µg/mL and above the test material precipitated in the cell culture medium. The precipitation did not interfere with the counting of the colonies.

RANGE-FINDING/SCREENING STUDIES:
In the range finding test, the substance was tested up to 5000 µg/plate in the absence and presence of S9 mix in S. typhimurium TA100 and E. coli WP2uvrA. No reduction of the bacterial lawn and no biologically relevant decrease in the number of revertants were observed. The highest concentration analysed was selected based on the solubility of the test substance in the cell culture medium.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : Refer to pdf document provided under 'Attached background material'.

Any other information on results incl. tables

Detailed result tables, incl. individual plate counts, for the dose range finding experiment and both mutagenicity assays are provided in the pdf document provided under 'Attached background material'. Please note that the registrant is authorised to use the study report for the purpose of the REACH registration despite the 'CONFIDENTIAL' stamp on each page.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative with and without metabolic activation