2-ethoxy-1,3-dimethylcyclohexane

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:

17-Mar-2014 to 22-apr-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

2-ethoxy-1,3-dimethylcyclohexane

EC Number:

944-610-3

Cas Number:

286472-48-8

Molecular formula:

C10H20O

IUPAC Name:

2-ethoxy-1,3-dimethylcyclohexane

Test material form:

liquid

Details on test material:

- Substance name as cited in test report: FRET 05-0293
- Phystical state: clear, colorless liquid
- Storage conditions: ambient temperature (15-25 °C), protected from light

Specific details on test material used for the study:

- Name of test material (as cited in study report): IFF 05-0293
- Substance type: Clear colourless liquid
- Physical state: Liquid
- Purity: 98.3%
- Batch/Lot number: RDRW23746
- Expiration date of the lot/batch: 31 January 2016

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 Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without S9-mix: 10, 33, 100, 333 and 1000 µg/plate
With S9-mix: 10, 33, 100, 333, 1000 and 3330 µg/plate

Experiment 2:
TA1535, TA1537, TA98 and TA100:
Without S9-mix: 10, 33, 100, 333 and 1000 µg/plate
With S9-mix: 10, 33, 100, 333, 1000 and 3330 µg/plate
WP2uvrA
Without and with S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate

Experiment 3:
TA1537
With S9-mix: 10, 33, 100, 333 and 1000 µg/plate
With S9-mix: 10, 33, 100, 333, 1000 and 3330 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Test compound was soluble in ethanol and ethanol has been 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:

methylmethanesulfonate

Remarks:

TA100, without S9

Controls 2

Positive control substance:

2-nitrofluorene

Remarks:

TA98, without S9

Controls 3

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

WP2uvrA, without S9

Controls 4

Positive control substance:

sodium azide

Remarks:

TA1535, without S9

Controls 5

Positive control substance:

other: 2-aminoanthracene

Remarks:

all strains: 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. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

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

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

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

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:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- . In tester strain TA100, toxicity was observed at dose levels of 333 μg/plate and upwards in the absence of S9-mix and at dose levels of 1000 μg/plate and upwards in the presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
TA1535: without S9: 100 µg/plate and above and with S9: 1000 µg/plate and above
TA1537: without S9: 100 µg/plate and above and with S9: 1000 µg/plate and above
TA98: without S9: 100 µg/plate and above and with S9: 1000 µg/plate and above
TA100: without S9: 333 µg/plate and above and with S9: 1000 µg/plate and above
WP2uvrA: No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

Any other information on results incl. tables

Since in tester strain TA1537 in the second experiment no toxicity or precipitate on the plates was observed in the presence of S9-mix, a third mutation experiment was performed with this strain in the presence of S9-mix.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

IFF 05-0293 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Evaluation of the mutagenic activity of IFF 05-0293 in the Salmonella typhimurium reverse mutation

assay and the Escherichia coli reverse mutation assay (with independent repeat).

IFF 05-0293 was tested in the Salmonella typhimurium reverse mutation assay with four histidinerequiring

strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the

Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli

(WP2uvrA). The test was performed in two independent experiments in the presence and absence of

S9-mix (rat liver S9-mix induced by Aroclor). An additional experiment was performed with tester strain

TA1537 in the presence of S9-mix.

The study procedures described in this report were based on the most recent OECD and EC

guidelines.

Batch RDRW23746 of IFF 05-0293 was a clear colourless liquid with a purity of 98.3%. The test

substance was dissolved in ethanol.

In the dose range finding test, IFF 05-0293 was tested up to concentrations of 5000 μg/plate in the

absence and presence of S9-mix in the strains TA100 and WP2uvrA. IFF 05-0293 did not precipitate

on the plates at this dose level. In tester strain TA100, toxicity was observed at dose levels of

333 μg/plate and upwards in the absence of S9-mix and at dose levels of 1000 μg/plate and upwards

in the presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels

tested. Results of this dose range finding test were reported as part of the first mutation assay.

Based on the results of the dose range finding test, IFF 05-0293 was tested in the first mutation assay

in tester strains TA1535, TA1537 and TA98 at concentration ranges of 10 to 1000 μg/plate and 10 to

3330 μg/plate in the absence and presence of 5% (v/v) S9-mix, respectively. In an independent repeat

of the assay with additional parameters, IFF 05-0293 was tested at concentration ranges of 10 to

1000 μg/plate and 33 to 3330 μg/plate in the absence and presence of 10% (v/v) S9-mix respectively

in tester strains TA1535, TA1537, TA98, TA100 and at concentration ranges of 100 to 5000 μg/plate in

tester strain WP2uvrA. Toxicity was observed in all tester strains, except in the tester strain WP2uvrA

and in tester strain TA1537 in the presence of S9-mix (second experiment).

Since in tester strain TA1537 in the second experiment no toxicity or precipitate on the plates was

observed in the presence of S9-mix, a third mutation experiment was performed with this strain in the

presence of 10% (v/v) S9-mix. IFF 05-0293 was tested in the third mutation assay at a concentration

range of 100 to 5000 μg/plate. Toxicity was observed at the top dose of 5000 μg/plate.

IFF 05-0293 did not induce a significant dose-related increase in the number of revertant (His+)

colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of

revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic

activation. These results were confirmed in independently repeated experiments.

The negative control values were within the laboratory historical control data ranges, except for

TA1535 in the presence of S9-mix (first experiment). However, since this value was just outside the

limit of the range, the validity of the test was considered to be not affected.

The 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 IFF 05-0293 is not mutagenic in the Salmonella

typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.