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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 2009
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-benzofuran-1,3-dione, addition product with 2-(2-hydroxyethoxy)ethanol, ethoxylated
Molecular formula:
Not applicable (UVCB substance)
2-benzofuran-1,3-dione, addition product with 2-(2-hydroxyethoxy)ethanol, ethoxylated
Test material form:
Specific details on test material used for the study:
Stability in the solvent was analytically proved.


Target gene:
mutant histidine gene
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-Mix from the liver of Aroclor 1254 induced male Sprague Dawley rats.
Test concentrations with justification for top dose:
plate incorporation assay: 0, 50, 158, 500, 1581, 5000 µg/plate with and without S9 mix
preincubation assay: 0, 50, 158, 500, 1581, 5000 µg/tube with and without S9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance formed clear colorless solutions in DMSO.
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
other: Na-azide (only TA 1535), nitrofurantoin (only TA 100), 4-nitro-1,2-phenylene diamine (TA 1537 and TA 98), mitomycin C (only TA 102 in plate incorporation assay), cumene hydroperoxide (only TA 102 in preincubation assay), 2-aminoanthracene.
The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, mitomycin C and cumene hydroperoxide were only used without S9 mix; the positive control 2-aminoanthracene was only used with S9 mix.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) for initial testing; independent repeat as preincubation testing (preincubation for 20 min. at 37 °C);
Testings for each strain and dose with and without S9 mix was performed in triplicate, which is also valid for solvent and positive controls.

- background growth
- mutant count: A toxic effect was assumed when there was a marked and dose-dependent reduction in the mutant count per plate, compared to the negative controls.
Evaluation criteria:
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgment.
In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.
no statistics perfomed; evaluation based on criteria mentioned above

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA 1535, TA 100, TA 1537, TA 98, TA 102
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
Untreated negative controls validity:
not applicable
Positive controls validity:
Additional information on results:
None of the five strains concerned showed in the plate incorporation test a dose-related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to the tests with and without S9 mix and was confirmed by the results of the preincubation trials.

- Precipitation: No precipitation of the test item was observed on the plates.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No indication of a bacteriotoxic effect of the test substance at doses of up to and including 5000 µg per plate.

Any other information on results incl. tables

see attached table 5.2 - Tablulated Summary of Data

Applicant's summary and conclusion

Executive summary:

The test substance was investigated in a bacterial reverse mutation test (Ames) according to OECD TG 471 on Salmonella typhimurium TA 1535, TA 100, TA, 1537, TA 98, and TA 102 without and with S9 mix. Based on the results in the initially performed plate incorporation as well as in the preincubation modification, performed as independent repeat, the substance is considered to be non-mutagenic in bacteria.