Acetic acid, 2,2-difluoro-2-[[2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy]-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2009-03-09 to 2009-10-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The experimental study on the supporting substance is guideline-conform under GLP without deviations.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

cC6O4 ammonium salt

IUPAC Name:

cC6O4 ammonium salt

Constituent 2

Reference substance name:

Acetic acid, 2,2-difluoro-2-[[2,2,4,5- tetrafluoro-5-(trifluoromethoxy)-1,3- dioxolan-4-yl]oxy]-, ammonium salt (1:1)

IUPAC Name:

Acetic acid, 2,2-difluoro-2-[[2,2,4,5- tetrafluoro-5-(trifluoromethoxy)-1,3- dioxolan-4-yl]oxy]-, ammonium salt (1:1)

Constituent 3

Reference substance name:

Reaction mass of ammonium difluoro {[(4S,5R)-2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetate, ammonium difluoro {[(4R,5S)-2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetate, ammonium difluoro {[(4S,5S)-2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetate and ammonium difluoro {[(4R,5R)-2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetate

EC Number:

682-238-0

Cas Number:

1190931-27-1

Molecular formula:

C6H4F9NO6

IUPAC Name:

Reaction mass of ammonium difluoro {[(4S,5R)-2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetate, ammonium difluoro {[(4R,5S)-2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetate, ammonium difluoro {[(4S,5S)-2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetate and ammonium difluoro {[(4R,5R)-2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetate

Details on test material:

- Name of test material (as cited in study report): cC6O4 cyclic (SOLUTION 16%)
- Molecular formula (if other than submission substance): C6H4F9NO6
- Molecular weight (if other than submission substance): 357
- Smiles notation (if other than submission substance): [NH4+].FC1(F)OC(F)(OC(F)(F)C([O-])=O)C(F)(OC(F)(F)F)O1
- InChl (if other than submission substance): no data
- Structural formula attached as image file (if other than submission substance): see Fig.1 (C6O4 Structural formula)
- Substance type: pure substance in water solution
- Physical state: Yellowish solution
- Analytical purity: not reported
- Impurities (identity and concentrations): NH4F=28 (mg/l), NH4Cl=601(mg/l), the organic impurities are dioxolan based and represent the 0.8% by mol on total mol of organic phase of sample.
- Purity test date: not reported
- Lot/batch No.: 150/28
- Expiration date of the lot/batch: 31 December 2020
- Stability under test conditions: not reported
- Storage condition of test material: room temperature

Method

Target gene:

Salmonella typhimuriumtester strains: histidine requirement
E. coli (WP2 uvrA): tryptophan requirement

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

n.a.

Additional strain / cell type characteristics:

other: rfa wall mutation (increasing of permeability to certain classes of chemicals), deficiency in DNA excision repair system (uvrB mutation). The strain is predominantly sensitive to base pair mutagens.

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

n.a.

Additional strain / cell type characteristics:

other: rfa wall mutation (increasing of permeability to certain classes of chemicals), deficiency in DNA excision repair system (uvrB mutation). The strain is sensitive to frameshift mutagens.

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

n.a.

Additional strain / cell type characteristics:

other: rfa wall mutation (increasing of permeability to certain classes of chemicals), deficiency in DNA excision repair system (uvrB mutation). The strain is sensitive to frameshift mutagens. Presece of pKM101 plasmid.

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

n.a.

Additional strain / cell type characteristics:

other: rfa wall mutation (increasing of permeability to certain classes of chemicals), deficiency in DNA excision repair system (uvrB mutation). The strain is predominantly sensitive to base pair mutagens. Presence of pKM101 plasmid.

Species / strain 5

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

n.a.

Additional strain / cell type characteristics:

other: Tester strain contains an uvrA DNA repair deficiency which enhance its sensitivity to some mutagenic compounds.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Toxicity test: 5000, 1580, 500, 158 and 50.0 µg/plate.
Assays for reverse mutation I and II: 5000, 2500, 1250, 625 and 313 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used for the test item: Distilled Water.

- Vehicle(s)/solvent(s) used for the positive controls:
Distilled water for Sodium azide, Methylmethanesulphonate (MMS).
DMSO for 9-Aminoacridine, 2-Nitrofluorene, 2-Aminoanthracene.

Controls

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO, Distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Sodium azide, 9-Aminoacridinee, 2-Nitrofluorene, 2-Aminoanthracene, Methylmethanesulphonate

Details on test system and experimental conditions:

Preliminary toxicity test
A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in the main assays. In these tests a wide range of dose levels of the test item, set at half-log intervals, were used. Treatments were performed both in the absence and presence of S9 metabolism using the plate incorporation method; a single plate was used at each test point and positive controls were not included. Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.

Main experiments
Two experiments were performed including negative and positive controls in the absence and presence of an S9 metabolising system. Three replicate plates were used at each test point.
In addition, plates were prepared to check the sterility of the test item solutions and the S9 mix, and dilutions of the bacterial cultures were plated on nutrient agar plates to establish the number of bacteria in the cultures.
The first experiment was performed using a plate-incorporation method.
The second experiment was performed using a pre-incubation method.



METHOD OF APPLICATION: in agar (plate incorporation), pre-incubation (in suspension)

DURATION
- Preincubation period: 30 min
- Exposure duration: approximately 72 hours
- Expression time (cells in growth medium): approximately 72 hours
- Selection time (if incubation with a selection agent): n.a.
- Fixation time (start of exposure up to fixation or harvest of cells): n.a.

SELECTION AGENT (mutation assays): n.a.
SPINDLE INHIBITOR (cytogenetic assays): n.a.
STAIN (for cytogenetic assays): n.a.

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: The estimated numbers of viable bacteria/plate (titre) fell in the range of 100 - 500 million for each strain.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.

OTHER EXAMINATIONS:
- Determination of polyploidy: n.a.
- Determination of endoreplication: n.a.
- Other:

Evaluation criteria:

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Statistics:

The regression analysis fits a regression line to the data by the least squares method, after square root transformation of the plate counts to satisfy normal distribution and homoscedasticity assumptions. The regression equation is expressed as: y = a + bx.
Regression lines are calculated using a minimum of the three lowest dose levels, and then including the further dose levels in turn. The correlation co-efficient (r), the value of students "t" statistic, and the p-value for the regression lines are also given.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Main Assay I: without pre-incubation. Test concentrations: 5000, 2500, 1250, 625 and 313 µg/plate.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Main Assay I: without pre-incubation. Test concentrations: 5000, 2500, 1250, 625 and 313 µg/plate.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Main Assay II: with pre-incubation. Test concentrations: 5000, 2500, 1250, 625 and 313 µg/plate.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity was observed both in absence and presence of S9 metabolic activation on tested strains TA 1535, TA 1537, TA 98 at dose levels of 2500 and 5000 µg/plate and on strain TA 100 at dose levels of 1250, 2500 and 5000 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Main Assay II: with pre-incubation. Test concentrations: 5000, 2500, 1250, 625 and 313 µg/plate.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity was observed at dose levels of 2500 and 5000 µg/plate, both in the absence and presence of S9 metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none reported
- Effects of osmolality: none reported
- Evaporation from medium: not reported
- Water solubility: no limitations reported
- Precipitation: No precipitation of the test item was noted at the end of the incubation period at any concentration.
- Other confounding effects: none reported

RANGE-FINDING/SCREENING STUDIES: A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in the main assays. In these tests a wide range of dose levels of the test item, set at half-log intervals, were used.

COMPARISON WITH HISTORICAL CONTROL DATA: Results show that mean plate counts for untreated and positive control plates fell within the normal historical range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Main Assay I (without ptre-incubation): using the plate incorporation method, no toxicity was observed at any dose level with any tester strain both in the absence and presence of S9 metabolic activation.
Main Assay II (with pre-incubation): Toxicity, as indicated by thinning of the background lawn and/or reduction in revertant numbers, was observed at higher dose levels with all tester strains, both in the absence and presence of S9 metabolic activation.

STERILITY
The sterility of the S9 mix and of the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

cC6O4 ammonium salt does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Executive summary:

In order to evaluate the genotoxic properties of F- DIOX acid it was deemed appropriate to use the Read Across approach

based on the experimental study performed on cC6O4 ammonium salt by virtue of similarity of chemical structure between F- DIOX acid and cC6O4 ammonium salt (CAS no: 1190931-27-1).

C6O4 ammonium salt is the carboxylic acid derivative of F-DIOX acid and therefore C6O4 ammonium salt differs from F-DIOX acid only for the presence of the cationic part NH4+.

Considering the similarity of chemical structure a similar biological behaviour is expected.

The test item cC6O4 ammonium salt was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone.

The test item cC6O4 ammonium salt (solution in sterile distilled water)

was assayed in the toxicity test at a maximum dose level of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. No toxicity was observed at any dose level with any tester strain.

In Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels expressed in terms of active ingredient: 5000, 2500, 1250, 625 and 313 µg/plate. No toxicity was observed at any dose level with any tester strain both in the absence and presence of S9 metabolic activation.

As no relevant increases in revertant numbers were observed at any concentration tested, a pre-incubation step was included for all treatments of Main Assay II. The test item was assayed at the same concentrations employed in Main Assay I. Toxicity, as indicated by thinning of the background lawn and/or reduction in revertant numbers, was observed at higher dose levels with all tester strains, both in the absence and presence of S9 metabolic activation. The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

It is concluded that the test item cC6O4 ammonium salt does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.