Cyclohexane, 1,4-bis(ethoxymethyl)-

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 study according to current guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(1r,4r)-1,4-bis(ethoxymethyl)cyclohexane; (1s,4s)-1,4-bis(ethoxymethyl)cyclohexane

EC Number:

700-868-7

Cas Number:

54889-63-3

Molecular formula:

C12 H24 O2

IUPAC Name:

(1r,4r)-1,4-bis(ethoxymethyl)cyclohexane; (1s,4s)-1,4-bis(ethoxymethyl)cyclohexane

Details on test material:

- Name of test material (as cited in study report): 1,4-bis(ethoxymethyl)-Cyclohexane
- Physical state: Liquid; colorless, clear
- Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions.
- Analytical purity: 99.6 corrected area-%
- Lot/batch No.: 10474-11-115
- Date of production: 26 Jul 2012
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed until 26 Jul 2014 as indicated by the sponsor, and the sponsor holds this responsibility.
- Storage condition of test material: Room temperature

Method

Target gene:

his, trp

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:

Liver S9 fraction of phenobarbital / β-naphthoflavone treated Wistar rats

Test concentrations with justification for top dose:

see "Details on test system and conditions"

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited insolubility of the test substance in ultrapure water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine (TA 1535, TA 100); 4-nitro-o-phenylenediamine (TA 98); 9-aminoacridine (TA1537)); 4-nitroquinoline-N-oxide (E coli). With S9 mix: 2-aminoanthracene (all strains tested)

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation test; preincubation test

1st Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1 000; 2 500 and 5 000 μg/plate
Type of test: Standard plate test with and without S9 mix
Number of plates: 3 test plates per dose or per control

2nd Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1 000; 2 500 and 5 000 μg/plate
Type of test: Preincubation test with and without S9 mix
Number of plates: 3 test plates per dose or per control
Reason: No mutagenicity was observed in the standard plate test.

3rd Experiment
Strains: TA 1535, TA 100, TA 1537 and TA 98
Doses: 0; 3.3; 10; 33; 100; 333 and 1 000 μg/plate
Type of test: Preincubation test with S9 mix
Number of plates: 3 test plates per dose or per control
Reason: Strong bacteriotoxicity was observed in the 2nd experiment.

4th Experiment
Strains: TA 1535
Doses: 0; 3.3; 10; 33; 100; 333 and 1 000 μg/plate
Type of test: Preincubation test with S9 mix
Number of plates: 3 test plates per dose or per control
Reason: Concerning the bacteriotoxicity, inconclusive values were observed
in the 3rd experiment.

DURATION
- Preincubation period: 20 minutes (preincubation test)

DETERMINATION OF CYTOTOXICITY
Toxicity is detected by a
• decrease in the number of revertants
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)
• reduction in the titer

Evaluation criteria:

Acceptance criteria
Generally, the experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 10^9 cells per mL were used.

Assessment criteria
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control data range under all experimental conditions in at least two experiments carried out independently of each other.

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

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 examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test substance precipitation was found with and without S9 mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ revertants, slight reduction in the titer) was observed in the standard plate test depending on the strain and test conditions from about 2 500 μg/plate onward.
In the preincubation assay strong bacteriotoxicity (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed depending on the strain and test conditions from about 100 μg/plate onward.
The inconclusive Data observed in the 3rd Experiment with tester strain TA 1535 was not confirmed in the repeat experiment and has to be considered as not relevant.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

According to the results of the present study, the test substance did not lead to a relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in several experiments carried out independently of each other

(standard plate test and preincubation assay).

Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study.

In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.

In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.
Thus, under the experimental conditions of this study, the test substance 1,4-bis(ethoxymethyl)-Cyclohexane is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.
The present data on genetic toxicity do not fulfill the criteria laid down in 67/548/EEC and regulation (EU) 1272/2008 and therefore, a non-classification is warranted.