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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 27, 2018 to September 24, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Dihexyl ether
EC Number:
203-987-8
EC Name:
Dihexyl ether
Cas Number:
112-58-3
Molecular formula:
C12H26O
IUPAC Name:
1-(hexyloxy)hexane
Test material form:
liquid

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction, derived from male Wistar rats, phenobarbital (80 mg/kg bw) / ß-naphthoflavone (100 mg/kg bw) induced for three consecutive days by oral route
Test concentrations with justification for top dose:
Pre-experiment: 0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5, 5.0 µL/plate

Experiment I:
TA98 with/without metabolic activation and TA100 with metabolic activation: 3.16, 10.0, 31.5, 100, 316, 1000, 2500, 5000 nl/plate
TA100, TA1535, TA 1537 and TA 201 without metabolic activation: 0.036, 0.0100, 0.316, 1.00, 3.16, 10, 31.6, 100, 316 nL/plate
TA1535, TA1537, TA 102 with metabolic activation: 1.00, 3.16, 10, 31.6, 100, 316, 1000 and 2500 nL/plate

Experiment II:

without metabolic activation: 0.0632, 0.200, 0.632, 2.00, 6.32, 20.0, 63.2 and 200 nL/plate
with metabolic activation: 0.632, 2.00, 6.32, 20.0, 63.2, 200, 632 and 2000 nL/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 2-Aminoanthacene: 2.5 µg/plate in DMSO for TA98, TA100, TA1535, TA 1537, TA102 with met. activ.; 10 µg/plate in DMSO for TA102 with met. activ. 4-nitro-o-phenylene-diamine: 10 µg/plate for TA98 without met. activ.; 40 µg/plate for TA1537 without met.act.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 plates

DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approx. ≤ 0.5 in relation to the solvent control

OTHER:
The properties of the S. typhimurium with regard to membrane permeability, ampicillin- and tetracycline-resistance as well as normal spontaneous mutation rates are checked regularly according to Ames et al..
The colonies were counted using a ProtoCOL counter (Meintrup DWS Laborgeräte GmbH, Germany). Tester strains with a low spontaneous mutation frequency (TA 1535 and TA 1537) were counted manually.
Evaluation criteria:
Evaluation of Mutagenicity:
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean
values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and E. coli WP2 uvrA the number of reversions is at least twice
as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
as compared to the reversion rate of the solvent control.
According to the OECD guidelines, the biological relevance of the results is the criterion for the
interpretation of results, a statistical evaluation of the results is not regarded as necessary.
A test item producing neither a dose related increase in the number of revertants nor a reproducible
biologically relevant positive response at any of the dose groups is considered to be non-mutagenic
in this system.
Criteria of validity:
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA98, TA100, TA102)
- the negative control plates (A. dest.) with and without S9 mix are within the historical control data ranges
-corresponding background growth on both negative control and test plates is observed.
- the positive controls show a distinct enhancement of revertant rates over the control plate.
- at least five different concentrations of each tester strain are analysable.
Statistics:
A statistical evaluation of the results was not regarded as necessary as the biological relevance of the results is the criterion for the interpretation of results (according to guideline).

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxic effects observed in exp. I at 31.6 nl/plate and higher without metab. activ. and at 1000 nl/plate and higher with metab. activ, In exp. II at 63.2 nl/plate and higher without metab. activ. and at 2000 nl/plate with metab. activ.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxic effects observed in exp. I at 31.6 nl/plate and higher without metab. activ. and at 1000 nl/plate and higher with metab. activ, In exp. II at 63.2 nl/plate and higher without metab. activ. and at 316 nl/plate and higher with metab. activ.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxic effects observed in exp. I at 31.6 nl/plate and higher without metab. activ. and at 1000 nl/plate and higher with metab. activ, In exp. II at 63.2 nl/plate and higher without metab. activ. and at 2000 nl/plate with metab. activ.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxic effects observed in exp. I at 10.0 nl/plate and higher without metab. activ. and at 100 nl/plate and higher with metab. activ, In exp. II at 6.32 nl/plate and higher without metab. activ. and at 2000 nl/plate with metab. activ.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxic effects observed in exp. I at 100 nl/plate and higher without metab. activ. and at 2500 nl/plate with metab. activ, In exp. II at 63.2 nl/plate and higher without metab. activ. and at 2000 nl/plate with metab. activ.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No recipitation of the test item was observed in any tester strains used in experiment I and II (with and without metabolic activation).

RANGE-FINDING/SCREENING STUDIES: The toxicity of the test item was determind with tester stains TA 98 and TA 100. Toxicity may be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

COMPARISON WITH HISTORICAL CONTROL DATA: The control plates with and without S9 mix were within the historical control data range.

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative with and without metabolic activation

The test substance was considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

The test item dihexyl ether was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I and experiment II) using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102.

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. Precipitation of the test item was not observed in any tester strains used in experiment I and II (with and without metabolic activation).

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with dihexyl ether at any concentration level tested, neither in the presence nor absence of metabolic activation in experiment I and II. The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the two independent experiments.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, dihexyl ether did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, dihexyl ether is considered to be non-mutagenic in this bacterial reverse mutation assay.