Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21. Jul. 1997
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
adopted 30. May 2008
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material: 1,3,2-dioxathiane 2,2-dioxide
- IUPAC name: 1,3,2-Dioxathiane 2,2-dioxide
- Molecular formula: C3H6O4S
- Molecular weight: 138.14 g/mol
- Substance type: Organic
- Physical state: Solid

Method

Species / strainopen allclose all
Species / strain / cell type:
other: Salmonella typhimurium TA97a
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
metabolic activation by microsomal enzymes (e.g., benzo(a)pyrene)
Test concentrations with justification for top dose:
5000 µg/plate
Vehicle / solvent:
Yes, Vehicle
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 4-Nitro-1,2-phenylene diamine, C6H7N3O2; CAS-No.: 99-56-9 and 2-Amino-anthracene, C14H11N; CAS-No.: 613-13-8
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension

DURATION
- Preincubation period: 8 hours at 37 ± 1 °C
- Exposure duration: First Experiment (48 hours at 37 ±1 °C)
- Expression time (cells in growth medium): 24 hours at 37 ±1 °C
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours at 37 ±1 °C

SELECTION AGENT (mutation assays): mutagenic substances: 4-Nitro-1,2-phenylene diamine (CAS#99-56-9), Sodium azide (CAS#26628-22-8), 2-Amino-anthracene(CAS#613-13-8) and Benzo-a-pyrene(CAS#50-32-8)

NUMBER OF REPLICATIONS: 3
Rationale for test conditions:
All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.
Evaluation criteria:
The determination of titre should give a number of at least 109 cells/mL

The determination values for the spontaneous revertants of the negative controls and positive controls (diagnostic mutagens) were within the historical control data ranges.
Statistics:
The confirmation tests of the genotype did not show any irregularities. The control of the titre was above the demanded value of 109 bacteria/mL. Nearly all of the means of all replicates of the spontaneous revertants (in negative and solvent controls) were within the range of the historical data of the test facility. All numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory and were increased in comparison with the negative controls, which demonstrated the mutagenic potential of the diagnostic mutagens.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
other: TA97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Based on the results of this study it is concluded that 1,3,2-Dioxathiane, 2,2-dioxide is mutagenic in the Salmonella typhimurium test strains TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.
Executive summary:

Two valid experiments were performed.

 

The study procedures described in this report were based on the most recent OECD and EC guidelines.

The test item1,3,2-Dioxathiane, 2,2-dioxidewas tested in theSalmonella typhimuriumreverse mutation assay with five strains ofSalmonella typhimurium(TA97a, TA98, TA100, TA102 and TA1535).

The test was performed in two experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation.

 

Experiment 1:

In the first experiment,the test item (dissolved in DMSO) was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test itemshowed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.

The results of this experiment showed that all tested concentrations showed an increase in the number of revertants in the tested strains TA98, TA100, TA102 and TA1535, in the presence and the absence of metabolic activation.

 

Based on the results of this study it is concluded that1,3,2-Dioxathiane, 2,2-dioxideis mutagenic in theSalmonella typhimuriumstrains TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.