1,4-dioxane-2,5-dione

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998-07-30 to 1998-09-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

adopted December 29, 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,4-dioxane-2,5-dione

EC Number:

207-954-9

EC Name:

1,4-dioxane-2,5-dione

Cas Number:

502-97-6

Molecular formula:

C4H4O4

IUPAC Name:

1,4-dioxane-2,5-dione

Test material form:

solid: particulate/powder

Method

Target gene:

Histidine locus (Salmonella typhimurium strains) and tryptophan locus (E. coli strain)

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:

S9 mix

Test concentrations with justification for top dose:

1st and 2nd test: 0, 62, 185, 556, 1667 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was chosen as vehicle because the test substance decomposes in water.

Controls

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: N-ethyl-N-nitrosourea (without metabolic acitivation); 2-aminoanthracene (with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: none
- Exposure duration: 3 days

NUMBER OF REPLICATIONS: Two experiments, all determinations in triplicate

DETERMINATION OF CYTOTOXICITY
- Cytotoxicity is defined as a reduction in the number of revertant colonies and/or a clearing of the background lawn of bacterial growth

Evaluation criteria:

The study is considered valid if the mean colony counts of the control values of the strains are within acceptable ranges and if the results of the positive controls meet the criteria for a positive response.
A response is considered to be positive if the mean number of revertant colonies on the test plates is increased two-fold or more compared to that on the vehicle control plates.
A test substance is considered to be mutagenic if a concentration-related increase or if a positive response reproducible in both assays is observed.
A test substance is considered to be not mutagenic in the bacterial gene mutation test if it produces neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points.

Statistics:

No statistical analysis is required.

Results and discussion

Test results

Key result

Species / strain:

other: S. typhimurium TA 98, TA 100, TA 1535, Ta1537 and E.coli WP2 uvrA

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

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A precipitate on the plates was observed in the first test only at 1667 µg/plate and higher.

COMPARISON WITH HISTORICAL CONTROL DATA:The mean number of his+ and trp+ revertant colonies of the negative controls were within the acceptable range, and the positive controls gave the expected increase in the mean number of revertant colonies

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test substance Glycolide was not toxic to any strain, as was evidenced by the absence of decrease in the mean number of revertant colonies

Applicant's summary and conclusion

Conclusions:

It is concluded that the results obtained with the test substance 1,4-Dioxane-2,5-dione (min 99.5%) in a reverse gene mutation assay in bacteria according to OECD guideline 471 (adopted July 21, 1997) and EU method B.13/14 (December 29, 1992) with the strains TA 1535, TA 1537, TA 98, TA 100 of S. typhimurium and E. coli  WP2 uvrA in the presence and absence of mammalian metabolic activation (S9 mix), indicate that 1,4-Dioxane-2,5-dione (Glycolide) was not mutagenic under the conditions employed in this study.