Indene

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:

19 Aug 2005 - 01 Sep 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21st July 1997

Deviations:

yes

Remarks:

See "Principles of method other than guideline"

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Testing Methods for New Chemical Substances etc. (Yakushokuhatsu No. 1121002, 21st November 2003; Seikyoku No.2, 13th November, 2003; Kampo Kihatsu No. 031121002)

Version / remarks:

2003

Deviations:

no

Principles of method if other than guideline:

Deviations:
OECD guide 471 line states that "2-Aminoanthracene should not be used as the sole indicator of the efficacy of the S9-mix. If 2-
aminoanthracene is used, each batch of S9 should also be characterised with a mutagen that requires
metabolic activation by microsomal enzymes, e.g., benzo(a)pyrene, dimethylbenzanthracene." In the study 2-Aminoanthracene was used as a sole indicitaor for the efficacy of the S9-mix, but the batch of S9 was not reported to have been further characterised with a mutagen that requires metabolic activation.
However, since a significant increase in revertants was seen for all strains, the deviation is not considered to affect the study integrity.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Indene

EC Number:

202-393-6

EC Name:

Indene

Cas Number:

95-13-6

Molecular formula:

C9H8

IUPAC Name:

indene

Test material form:

liquid

Details on test material:

Identification: Indene

Method

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

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:

Rat liver S9-mix induced by a combination of phenobarbital and 5,6-Benzoflavone

Test concentrations with justification for top dose:

Experiment 1: Dose finding study
Preliminary test (without and with S9): TA1535, TA1537, TA98 TA100 and WP2uvrA: 8.19, 20.5, 51.2 , 128, 320, 800, 2000 and 5000 µg/plate

Main study: TA1535, TA1537, TA98 TA100 and WP2uvrA
Without and with S9-mix: 6.25, 12.5, 25.0, 50.0, 100, 200, 400 µg/plate

The highest concentration in the main study was chosen to be 400 µg/plate as in the dose finding study bacterial growth was inhibited at 320 µg/plate.

Vehicle / solvent:

- solvent used: DMSO
- Justification for choice of solvent:
The test substance is easily soluble in DMSO and DMSO is accepted and approved by authorities and international guidelines

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide

Remarks:

without S9 0.01 ug/plate for TA100 in DMSO for TA100

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide

Remarks:

Without S9 0.1 ug/plate in DMSO for TA98

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 80 ug/plate in DMSO for TA 1537

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 , 0.5 µg/plate in water for TA1535

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide

Remarks:

without S9 0.1 µg/plate in DMSO for WP2uvrA

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene in DMSO for all tester strains

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain.
- One dose finding study and one main study were performed.

NUMBER OF CELLS EVALUATED:
100uL of stock solution added on plate, approx 1-4x10E8 cells.


OTHER EXAMINATIONS:
- Microbial growth inhibtion was determined
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

The experimental results were judged positive when the number of revertant colonies was twice or more that of the negative control and showed a dose-relationship or reproducibility.

Statistics:

A statistical analysis was not used for evaluation.

Results and discussion

Test results

Test results 1

Key result

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation in dose finding assay: At the start of treatment, white turbid medium was observed at 800 ug/plate or more dose levels in presence and absence of S9 mix. In addition, white oil droplet-like precipitations were observed at 5000 ug/plate in absence of S9 mix and white powder-like precipitations were observed at 2000 ug/plate or more levels in presence of S9 mix.
At the colony counting, white powder-like precipitations were observed at 800 ug/plate or more dose levels in presence of S9 mix.

RANGE-FINDING/SCREENING STUDIES:
- Microbial growth inhibition was found at 320 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The mean number of revertant colonies in both the negative and positive controls were within the ranges based on the historical data as included in the report.

Applicant's summary and conclusion

Conclusions:

In an AMES test, performed according to OECD guideline 471 and GLP principles, Indene was found not to be mutagenic with or without metabolic activation.

Executive summary:

An AMES test was performed according to OECD guideline 471 and GLP principles. All bacterial strains showed negative responses up to and at the dose that inhibited bacterial growth (320 ug/plate), i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. Precipitation of the test substance was observed in dose levels above 800 ug/plate. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that Indene is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.