Reaction mass of potassium difluoro{[2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetate with potassiumfluoride and potassium carbonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

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:

5 January 2010 to 14 July 2010

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:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene 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 ammonium salt (dry salt)
- 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
- Physical state: solid
- Analytical purity: 97.5 %
- Impurities (identity and concentrations): not reported
- Composition of test material, percentage of components: not reported
- Isomers composition: not reported
- Purity test date: not reported
- Lot/batch No.: 156/39
- Expiration date of the lot/batch: December 31, 2020
- Stability under test conditions: not reported
- Storage condition of test material: In the refrigerator (+2 to +8 °C)

Method

Target gene:

TK+/-

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Prior to mutagenicity testing the amount of spontaneous mutants is reduced by growing the cells for one day in RPMI 1640-HAT medium supplemented with:
hypoxanthine 1.0×10-4 M aminopterin 2.0×10-7 M thymidine 1.6×10-5 M
The incubation of the cells in HAT-medium is followed by a recovery period of 2 days in RPMI 1640 medium containing:
hypoxanthine 1.0×10-4 M thymidine 1.6×10-5 M
After this incubation the L5178Y cells are returned to normal RPMI 1640 medium (complete culture medium).

Large stocks of the cleansed L5178Y cell line are stored in liquid nitrogen in the cell bank of Harlan CCR allowing the repeated use of the same cell culture batch in many experiments. Before freezing, each batch was screened for mycoplasma contamination and checked for karyotype stability. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.
Thawed stock cultures are propagated in plastic flasks (Greiner, 72632 Frickenhausen, Germany) in RPMI 1640 complete culture medium. The cells are subcultured two times prior to treatment. The cell cultures are incubated at 37 ± 1.5°C in a humidified atmosphere with 4.5 % carbon dioxide and 95.5 % ambient air.

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

Experiment I:
without S9 mix (4 h treatment) : 56.9; 113.8; 227.5; and 455 μg/mL
with S9 mix (4 h treatment) : 56.9; 113.8; 227.5; 455; and 910 μg/mL

Experiment II:
without S9 mix (24 h treatment) : 28.1; 56.3; 112.5; 225; and 337.5 μg/mL
with S9 mix (4 h treatment) : 56.3; 112.5; 225; 450; and 675 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Deionised water
The test item was dissolved in deionised water on the day of the experiment (immediately before treatment).
The final concentration of deionised water in the culture medium was 10 % v/v.
Concurrent solvent controls (deionised water) were performed.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Referred to experiments I and II without metabolic activation

Migrated to IUCLID6: Supplier: Sigma, 82024 Taufkirchen, Germany Charge: 76296KJ Expiration Date: May 2010 Purity: > 99% Dissolved in: Nutrient medium Final Concentration: 19.5 μg/mL = 0.18 mM

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Referred to experiments I and II with metabolic activation.

Migrated to IUCLID6: Supplier: Aldrich Chemie, 89555 Steinheim, Germany Charge: 097K1311 Expiration Date: March 2011 Purity: ≥ 98 % Dissolved in: 0.9 % saline Final Concentration: 3.0 μg/mL = 10

Details on test system and experimental conditions:

PRE-TEST ON TOXICITY:
A pre-test was performed in order to determine the concentration range of the mutagenicity experiments. Both, pH value and osmolarity were determined at the maximal concentration of the test item and in the solvent control without metabolic activation.
1x107 cells (3x106 cells at the beginning of 24 h treatment) were exposed to each concentration of the test item for 4 and 24 hours without and 4 hours with metabolic activation. During the 4 h treatment period the serum concentration was reduced from 15 % to 3 %. Following treatment the cells were washed twice by centrifugation (425 g, 10 min) and resuspended in "saline G". Subsequently the cells were resuspended in 30 mL complete culture medium for a 2-day growth period. The cell density was determined immediately after treatment and at each day of the growth period and adjusted to 3x105 cells/mL, if necessary. The relative suspension growth (RSG) of the treated cell cultures was calculated at the end of the growth period according to the method of Clive and Spector.

DOSE SELECTION
According to the results of the pre-test at least four adequate concentrations were chosen for the mutation experiments.
The highest concentration should be 10 mM, but not higher than 5 mg/mL, unless limited by the solubility or toxicity of the test item.
RSG (Relative Suspension Growth) or RTG (Relative Total Growth) values (main experiment) below 50 % are considered toxic. In case of toxic effects, the highest test item concentration of the main experiment should reduce the RSG or RTG value to approximately 10 - 20 %.
The pre-experiment was performed in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Test item concentrations between 28.4 μg/mL and 3640 μg/mL were used. The highest concentration in the pre-experiment was chosen with regard to the purity (97.5 %) and the molecular weight (357 g/mol) of the test item. Strong toxic effects were observed at 1820 μg/mL and above in the absence of metabolic activation (4 h treatment). In the presence of metabolic activation (4 hours treatment) toxic effects occurred at 455 μg/mL and above. Following continuous treatment (24 hours) strong toxic effects were determined at 227.5 μg/mL and above.
The test medium was checked for precipitation at the end of each treatment period (4 or 24 hours) before the test item was removed. After 4 hours treatment precipitation of the test item was observed at 910 μg/mL and above in the presence and absence of metabolic activation. Following continuous treatment no precipitation was noted.
There was no relevant shift of the pH value or the osmolarity of the medium even at the maximum concentration of the pre-experiment.
The dose range of the main experiments was limited by the occurrence of toxicity and precipitation. The individual concentrations of the first experiment with and without metabolic activation and the second experiment without metabolic activation were generally spaced by a factor of 2.0. A narrower spacing was used in the upper concentration range to cover the limit of solubility or cytotoxic effects more closely.
Following the expression phase of 48 hours the cultures at the concentrations of 910 and 1365 μg/mL (printed in bold letters) in experiment I without metabolic activation and at 1365 μg/mL with metabolic activation were not analysed due to exceedingly severe toxic effects. In the second experiment the cultures at 450 μg/mL without metabolic activation and 675 μg/mL (culture I) and 900 μg/mL with metabolic activation were not continued for the same reason.

EXPERIMENT PERFORMANCE
In the mutation experiment 1×107 (3x106 during 24 h exposure) cells/flask (80 cm2 flasks) suspended in 10 ml RPMI medium with 3 % horse serum (15 % horse serum during 24 h exposure) were exposed to various concentrations of the test item either in the presence or absence of metabolic activation. After 4 h (24 h in the second experiment) the test item was removed by centrifugation (425 x g, 10 min) and the cells were washed twice with "saline G". Subsequently the cells were resuspended in 30 ml complete culture medium and incubated for an expression and growth period of 48 h.
The cell density was determined each day and adjusted to 3×105 cells/ml, if necessary. The relative suspension growth (RSG) of the treated cell cultures was calculated by the day 1 fold-increase in cell number multiplied by the day 2 fold-increase in cell number according to the method of Clive and Spector.
After the expression period the cultures were selected. Cells from each experimental group were seeded into 2 microtiter plates so that each well contained approximately 4×103 cells in selective medium (see below) with TFT (Serva, 69042 Heidelberg, Germany). The viability (cloning efficiency) was determined by seeding about 2 cells per well into microtiter plates (same medium without TFT). The plates were incubated at 37°± 1.5 °C in 4.5 % CO2/95.5 % water saturated air for 10 - 15 days. Then the plates were evaluated.

SiZE DISTRIBUTION OF THE COLONIES
Colonies were counted manually. In accordance with their size the colonies were classified into two groups. The colony size distribution was determined in the controls and at all concentrations of the test item. Criteria to determine colony size were the absolute size of the colony (more than 1/3 of a well for large colonies) and the optical density of the colonies (the optical density of the small colonies is generally higher than the optical density of the large ones).

Evaluation criteria:

A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 106 cells above the corresponding solvent control.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
However, in the evaluation of the test results the historical variability of the mutation rates in the solvent controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth is less than 10 % of the vehicle control unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance were considered together.

Results and discussion

Test results

Test results 1

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at: 227.5 μg/mL (experiment I), 225 μg/mL (experiment II)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at: 227.5 μg/mL (experiment I), 225 μg/mL (experiment II)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Precipitation of the test item visible to the naked eye was noted at 455 μg/mL and above in the first experiment with metabolic activation and at 450 μg/mL and above in the second experiment with metabolic activation.
Relevant toxic effects indicated by a relative total growth of less than 50 % of relative total growth were observed in experiment I starting at 227.5 μg/mL in the presence and absence of metabolic activation. In the second experiment toxic effects as described above occurred at 225 μg/mL and above with and without metabolic activation. The data generated in the second experiment without metabolic activation at 337.5 μg/mL were not judged as acceptable since the RTG dropped far below the 10% limit in both parallel cultures. The same was true for the data generated in the second experiment with metabolic activation at 675 μg/mL. The RTG of the second culture was only 2.6, the first culture was not analysable at all due to exceedingly severe cytotoxic effects.
No substantial and reproducible dose dependent increase of the mutation frequency was observed in both experiments up to the maximum concentration with and without metabolic activation. The mutation frequency exceeded the threshold of 126 above each solvent control in the first culture of the first experiment without metabolic activation at 227.5 and 455.0 μg/mL. In the second culture of the first experiment with metabolic activation the threshold was exceeded at precipitating concentrations of 450 and 910 μg/mL. However, this increase was not reproduced in the parallel culture under identical conditions and consequently judged as artefact. In the second experiment the threshold of 126 above each solvent control count was again exceeded in one of the parallel cultures at a precipitating concentration of 450 μg/mL with metabolic activation. Again, no comparable increase was noted in the parallel culture under identical conditions so this isolated increase was judged as precipitation artefact. Precipitation is a problem in the mouse lymphoma assay since L5178Y cells grow in suspension. The test item is removed by centrifugation at the end of treatment. At precipitating concentrations the test item can not completely be removed at the intended end of treatment since the precipitate sediments together with the cells upon centrifugation. Arbitrary amounts of the test item are carried over to the next steps of the assay leading to irreproducible artefacts.

Remarks on result:

other: strain/cell type: mouse lymphoma L5178Y cells

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

In this study the range of the solvent controls was from 124 up to 196 mutant colonies per 106 cells; the range of the groups treated with the test item was from 124 up to 764 mutant colonies per 106 cells. The solvent controls exceeded the recommended upper limit of 170 per 106 cells somewhat in the presence of metabolic activation and in one of both cultures following long term treatment for 24 hours. However, the data are judged as acceptable since the IWGT recommendations published in 2003 stated an upper limit of 200 colonies per 106 cells and all solvent controls remained within the historical control range. MMS (19.5 μg/mL in experiment I and 13.0 μg/mL in experiment II) and CPA (3.0 and 4.5 μg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies at acceptable levels of toxicity with at least one of the concentrations of the controls.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

It can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.
Therefore, cC6O4 AMMONIUM SALT is considered to be non-mutagenic in this mouse lymphoma assay.
Considering the similarity of chemical structure between F- Diox potassium salt and cC6O4 ammonium salt, it can be concluded that the same bahaviour for F- Diox potassium salt is expected.

Executive summary:

In order to evaluate the genotoxic properties of F- Diox potassium salt 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 potassium salt and cC6O4 ammonium salt (CAS no: 1190931-27-1).

 

F-Diox potassium salt and cC6O4 ammonium salt are two salts of the same carboxylic acid, they differ only for the cationic part (K+ in F-Diox potassium salt and NH4+ in cC6O4 ammonium salt). Considering the similarity of chemical structure a similar biological behaviour isexpected.

The study was performed to investigate the potential of cC6O4 AMMONIUM SALT to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation. The highest concentration (3640 μg/mL) applied in the pre-experiment was chosen with regard to the molecular weight of the test item corresponding to a molar concentration of about 10 mM. The concentration range of the main experiments was limited by cytotoxic effects and precipitation of the test item. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Therefore, cC6O4 AMMONIUM SALT is considered to be non-mutagenic in this mouse lymphoma assay.

Considering the similarity of chemical structure between F- Diox potassium salt and cC6O4 ammonium salt, it can be concluded that the same bahaviour for F- Diox potassium salt is expected.