Hexafluoropropene, oxidized, oligomers, reduced, fluorinated

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:

30-APR-1999 to 27-SEP-1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD Guideline-conform study conducted under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

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:

Tetrafluoroethylene, oxidized, oligomers, reduced, decarboxylated

EC Number:

500-539-6

EC Name:

Tetrafluoroethylene, oxidized, oligomers, reduced, decarboxylated

Cas Number:

161075-02-1

Molecular formula:

CF2H-O-(CF2-CF2-O)m-(CF2-O)n-CF2H

Test material form:

liquid

Details on test material:

Purity: > 99%

Method

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 pKM 101

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 fraction

Type and composition of metabolic activation system:
- source of S9: prepared at the facility
- method of preparation of S9 mix: the liver S9 fraction was prepared from 10 Sprague-Dawley rats induced with Aroclor 1254 (single ip injection at 500 mg/kg bw).
- concentration or volume of S9 mix and S9 in the final culture medium : 10% v/v (0.1 ml) for test 1; 30% v/v (0.3 ml) for test 2
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): the study included a sterility check with and without S9; the metabolic activation system was assess using the positive controls Benzo[a]pyrene and 2-aminoanthracene.

Test concentrations with justification for top dose:

5, 15, 50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

yes

Remarks:

untreated plates

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

benzo(a)pyrene

furylfuramide

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added to a 10-hour bacterial culture (at least 10E9 cells/ml) prior to plating (preincubation procedure)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 30 min at 37°C
- Exposure duration/duration of treatment: plates incubated for 72 hours at 37°C


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICITY
- revertant colonies per plate, triplicates.

METHOD OF APPLICATION: pre-incubation procedure in both assays
0.1 ml of the test article or solvent control solutions was placed into sterile test tubes containing an aliquot of 0.1 ml of a 10 hour bacterial culture and 0.5 ml S9 mix, or 0.5 ml sodium orthophosphate buffer (pH 7.4). The vial caps were sealed tightly. The vials were incubated at 37°C for 30 minutes with shaking. An aliquot of 2 ml molten agar containing 0.5 mM histidine/biotin/tryptophan was added to each vial. The mixture was thoroughly shaken and poured onto plates containing 25 ml minimal agar.
Three plates were prepared per test concentration, with or without metabolic activation.
Plates were also prepared without the addition of bacteria, in order to assess the sterility of the test substance, S9 mix and sodium orthophosphate buffer.
The test plates were incubated at 37 °C for ca. 72 hours.
The tubes and plates were suitably identified.
After the incubation period, the appearance of the background bacterial lawn was examined and the revertant colonies per plate were counted using a Domino automated colony counter.


A second test was conducted with modification of the S9 fraction content of the S9 mix, increased to 30%.

Evaluation criteria:

The test article is considered positive:
- if the increase in the number of revertant colonies is at least twice the concurrent solvent controls, with some evidence of positive dose-relationship, in 2 separate experiments, with any bacterial strain, either with or without S9 mix.


The test article is considered to produce no evidence of mutagenic activity:
- if treatment with the test substance does not produce reproducible increases of at least 1.5-times the concurrent solvent controls in either mutation test.

If the results obtained failed to satisfy the criteria for a clear "positive" or "negative" response as stated above, additional testing may be performed. Should an increase in revertant colony numbers then be observed which satisfy "positive" response, the substance is considered to show evidence of mutagenic activity in this test system.
If no clear "positive" response can be obtained, the test data may be subject to analysis to determine the statistical significance of any increases in revertant colony numbers, using usually the analysis of varia,ce followed by the Dunett's test.

Statistics:

The mean and the standard deviation were calculated for numbers of revertants at each concentration.
No statistical analysis was conducted in this study.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

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

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

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

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

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

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

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

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

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

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In both tests, there was no substantial increase in the number of revertant colonies in comparison with the negative controls in any of the tester strains following exposure to the test substance at any concentration up to the limit concentration of 5000 g/plate, in either the presence or absence of metabolic activation.
No visible thinning of the background lawn of non-revertant cells was obtained following exposure to the test substance.

The sterility controls confirmed the absence of microbial contamination.
The mean revertants colony counts for the solvent controls were within the expected ranges.
The positive control substances induced substantial increases in the number of revertant colonies, confirming the sensitivity of the system and activity of the metabolic activation.

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the test substance H GALDEN did not show any mutagenic activity in the reverse mutation assay up to the standard limit concentration of 5000 g/plate in Salmonella typhimurium (TA98, TA100, TA1535, TA1537 strains) and in Escherichia coli (WP2uvrA pKM101 strain), both with and without metabolic activation.

Executive summary:

To assess the potential mutagenic activity of Ethene, tetrafluoro-, oxidized, polymd., reduced, decarboxylated a reverse mutation assay was performed according to the OECD test guideline No. 471, EU Method B.13/14 and in compliance with GLP. Bacterial strains of Salmonella typhimurium (TA98, TA 100, TA 1535, and TA 1537) and Escherichia coli (WP2uvrA pKM101) were exposed to the test substance dissolved in dimethyl sulfoxide, at the following concentrations both in the presence and absence of metabolic activation system (S9-mix).

- test 1 (which served as range finding assay): 5, 15, 50, 150, 500, 1500, 5000 µg/plate, with and without S9.

- test 2 (repeat assay): 50, 150, 500, 1500, 5000 µg/plate with and without S9.

Both assays were conducted using the pre-incubation method. The conditions of the test procedure were modified in the confirmation assay using 30% (v/v) S9 (test 2), instead of 10% (v/v) (test 1).

There was no precipitate or visible thinning of the background lawn.

The sterility controls confirmed the absence of microbial contamination.

Acceptable responses were obtained for the negative and strain-specific positive controls indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

In both independent test, there was no substantial increase in the number of revertant colonies in comparison with the negative controls in any of the tester strains following exposure to the test substance at any concentration up to the limit concentration of 5000 g/plate, in either the presence or absence of metabolic activation.

Based on the results of this study, it is concluded that Ethene, tetrafluoro-, oxidized, polymd., reduced, decarboxylated (H GALDEN)

is not mutagenic in the reverse mutation assay with Salmonella typhimurium and Escherichia coli.