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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:
27 May - 5 June, 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Ditetrahydrofurylpropane
- Lot/batch No.: 6-7B09
- Analytical purity: 99.7%
- Substance type: Clear colourless liquid
- Physical state: liquid
- Purity test date: 09 February 2007
- Expiration date of the lot/batch: 09 February 2010
- Storage condition of test material: At room temperature in the dark
- Stability under storage conditions: Stable

Method

Target gene:
Histidine gene in S. typhimurium
Tryptophan gene in E. coli
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and ß-naphthoflavone induced rat liver S9 mix
Test concentrations with justification for top dose:
Dose range finding test: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
First and second mutation test: 100, 333, 1000, 3330 and 5000 µg/plate
The S9-mix contained 5% (v/v) S9 fraction in the dose range finding test and the first mutation test when tested in the presence of S9-mix.
The S9-mix contained 10% (v/v) S9 fraction in the second mutation test when tested in the presence of S9-mix.
Vehicle / solvent:
dimethyl sulfoxide
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the substance is soluble in DMSO and stable for at least 96 hours

Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
for TA1535 without S9, 5 µg/plate in saline
Positive control substance:
sodium azide
Positive controls:
yes
Remarks:
for TA1537 without S9, 60 µg/plate in Milli-Q water
Positive control substance:
9-aminoacridine
Positive controls:
yes
Remarks:
for TA98 without S9, 10 µg/plate in DMSO
Positive control substance:
2-nitrofluorene
Positive controls:
yes
Remarks:
for TA100 without S9, 650 µg/plate in DMSO
Positive control substance:
methylmethanesulfonate
Positive controls:
yes
Remarks:
for WP2uvrA without S9, 10 µg/plate in DMSO
Positive control substance:
4-nitroquinoline-N-oxide
Positive controls:
yes
Remarks:
for all strains with S9 (5% and 10%)
Positive control substance:
other: 2-aminoanthracene
Remarks:
Concentrations range from 1-10 µg/plate in DMSO
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS:
At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain.
The test substance was tested both in the absence and presence of S9-mix in each strain, in two independent experiments.

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:
- Other: precipitation of the test substance
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 (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
b) In case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Results and discussion

Test resultsopen allclose all
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:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: No reduction of the bacterial background lawn and no decrease or increase in the number of revertants were observed up to 5000 µg/plate.

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.

Applicant's summary and conclusion

Conclusions:
An AMES test was performed according to OECD/EC guidelines and GLP principles. All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.Based on these findings it is concluded that Ditetrahydrofurylpropane is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.