1,1-dimethoxyoctane

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:

13 December 2013 - 13 March 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

In accordance with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

yes

Remarks:

For the confirmatory assay only two, in stead of three, non-toxic dose levels were available for tester strains TA100 with and without S9, and TA1537 without S9. The deviation did not affect the integrity or interpretability of the results of the study.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

1,1-dimethoxyoctane

EC Number:

233-018-4

EC Name:

1,1-dimethoxyoctane

Cas Number:

10022-28-3

Molecular formula:

C10H22O2

IUPAC Name:

1,1-dimethoxyoctane

Method

Target gene:

Histidine (S. typhimurium) and tryptophan (E. coli)

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 homogenate (S9-mix) induced by Aroclor 1254

Test concentrations with justification for top dose:

- Initial mutagenicity assay: 5, 16, 50, 160, 500, 1600 and 5000 μg/plate (all strains with and without S9-mix)
- Independent confirmatory assay: 157, 313, 625, 1250 and 2500 µg/plate (all strains with S9-mix) and 50, 100, 200, 400 and 800 (all strains without S9-mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

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

benzo(a)pyrene

other: ICR-191: TA1537 (without S9) / 2-aminoanthracene: TA100, TA1535, TA1537 & WP2uvrA (with S9)

Remarks:

All positive control articles were dissolved in dimethyl sulfoxide (DMSO) with the exception of sodium azide which was dissolved in deionized water.

Details on test system and experimental conditions:

METHOD OF APPLICATION: Plate incorporation methodology

DURATION
- Exposure duration: 52 ± 4 hours at ca. 37 ± 2°C

NUMBER OF REPLICATIONS: All determinations were made in triplicate.

DETERMINATION OF CYTOTOXICITY
- Method: Macroscopic and microscopic (using a dissecting microscope) evaluation of the condition of the bacterial background lawn.
- Any supplementary information relevant to cytotoxicity: Evidence of cytotoxicity was scored relative to the vehicle control plate and was recorded along with the revertant counts for all plates at that dose level.

Evaluation criteria:

The test substance is considered to be positive in the bacterial gene mutation test if the mean number of revertant colonies on the test plates was increased in a dose-related manner, that is =>2-fold vehicle control values for tester strains TA98, TA100, and WP2uvrA, or =>3-fold vehicle control values for tester strains TA1535 and TA1537. In addition, any response should be reproducible. A test substance was considered to be negative in the bacterial gene mutation test if no dose-dependent, =>2-fold or =>3-fold increases was observed in tester strains TA98, TA100, and WP2uvrA, or TA1535 and TA1537, respectively.

Statistics:

No statistical analysis was performed.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

other: S. Typhimurium TA98, TA100, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

With S9-mix: toxicity was seen at ≥1250 μg/plate in all strains except TA100 and TA1537 (≥625 μg/plate and at 2500 μg/plate, resp.). Without S9-mix: toxicity was seen at ≥400 μg/plate in all strains except TA100 and TA1537 (toxicity at ≥200 μg/plate).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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

Remarks:

Toxicity was observed at => 1250 μg/plate in the presence of S9, and at => 400 μg/plate in the absence of S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

other: S. Typhimurium TA98, TA100, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the presence of S9-mix, cytotoxicity was observed at a concentration of =>1600 μg/plate in all Salmonella tester strains. In the absence of S9, toxicity was observed at =>500 μg/plate in all Salmonella tester strains.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

E. coli WP2 uvr A

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:

not examined

Positive controls validity:

valid

Additional information on results:

The study comprised two studies; an initial mutagenicity test and a confirmatory mutagenicity assay.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed in both experiments

HISTORICAL CONTROL DATA
- Positive historical control data: All positive controls were within the acceptance limits as specified in the study report.
- Negative (vehicle) historical control data: All negative controls were within the acceptance limits as specified in the study report.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
During the initial mutagenicity test none of the concentrations tested showed an increase in the number of revertants for the Salmonella typhimurium strains and Escherichia coli strain. However, reduced background lawn growth was observed in S. typhimurium strains in cultures without S9 at 500 µg/plate and in cultures with or without S9 at 1600 µg/plate and 5000 µg/plate. No reduced background was observed in E. coli strain, with or without S9. Based on the results of the initial test, a confirmatory assay (also plate incorporation methodology) was performed. Again no increase in the number of revertants in any strain at any concentration. Reduced background lawn growth was observed with S9 in strain TA100 at 625 µg/plate, at 1250 µg/plate in TA98, TA100, and TA1535 and in all salmonella typhimurium strains at 2500µg/plate. Without S9 reduced background lawn growth was observed at 200 µg/plate in strains TA100 and TA1537 and in all Salmonella typhimurium strains at 400 µg/plate and 800 µg/plate. Reduced background lawn growth was also observed in the E. coli strain with S9 from 1250 µg/plate and without S9 from 400 µg/plate. Per protocol, a minimum of three non-toxic dose levels will be required for a valid assay; however, for the confirmatory assay only two non-toxic dose levels were available for tester strains TA100 with and without S9, and TA1537 without S9. The study deviation, as was stated in the report, neither affected the overall interpretation of study findings nor compromised the integrity of the study.

Remarks on result:

other: Results are for confirmatory mutagenicity assay

Applicant's summary and conclusion

Conclusions:

Under the conditions of this test, the test substance was considered to be non-mutagenic and therefore the substance does not need to be classified as mutagenic in accordance with the criteria outlined in Annex 1 of the CLP Regulation (1272/2008/EC) and its amendments.

Executive summary:

The test substance was examined for its possible mutagenic activity in the bacterial reverse mutation assay, performed in accordance with OECD TG 471 and GLP. In this assay S. typhimurium strains TA1535, TA1537, TA98, TA100 and the E. coli strain WP2uvrA were used, in the absence and presence of metabolic activation (S9-mix). The test article was evaluated in an initial mutagenicity assay (plate incorporation method) in all strains at doses of 5, 16, 50, 160, 500, 1600, and 5000 µg/plate with and without S9. An independent confirmatory assay (plate incorporation method) was subsequently performed in all strains at dose levels of 157, 313, 625, 1250 and 2500 µg/plate with S9 and at dose levels of 50.0, 100, 200, 400, and 800 µg/plate without S9. Positive and vehicle controls were evaluated concurrently, and all test and control articles were evaluated in triplicate plates. In all strains, the positive and negative controls gave the expected increase in the mean numbers of revertant colonies. Therefore, both experiments were considered valid. In the initial mutagenicity assay no cytotoxicity was observed in WP2uvrA, at any tested dose level in the presence or absence of S9. However, cytotoxicity was observed in all Salmonella strains at ≥1600μg/plate in the presence of S9. In the absence of S9, toxicity was observed at ≥500μg/plate in all Salmonella tester strains. Based on the results of the initial assay, a confirmatory test was performed. In the presence of S9, toxicity was observed at ≥1250μg/plate in all tester strains except TA100 and TA1537. In TA100, toxicity was observed at ≥625μg/plate, whereas in TA1537, toxicity was observed only at 2500μg/plate. In the absence of S9, toxicity was observed at ≥400μg/plate in all tester strains except TA100 and TA1537, where toxicity was observed at ≥200μg/plate. This resulted in only two non-toxic dose levels for TA100 (with and without S9) and TA1537 (without S9), in the confirmatory assay. This is however considered not to influence the integrity and interpretability of the study. In both tests, in all strains tested, in both the absence and presence of S9-mix, the test substance did not induce a more than 2-fold and/or dose-related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed of the negative control. It is therefore concluded that the test substance is not mutagenic under the conditions of this test.