Reaction mass of (3E)-1,1,1,2,2,3,4,5,6,6,7,7,7-tridecafluoro-5-methoxyhept-3-ene and (2E)-1,1,1,2,3,4,5,5,6,6,7,7,7-tridecafluoro-4-methoxyhept-2-ene and (3E)-1,1,1,2,2,4,5,5,6,6,7,7,7-tridecafluoro-3-methoxyhept-3-ene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Remarks:

Conducted according to guideline in effect at time of study conduct

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

Conducted according to guideline in effect at time of study conduct

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

Conducted according to guideline in effect at time of study conduct

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(2E)-1,1,1,2,3,4,5,5,6,6,7,7,7-tridecafluoro-4-methoxyhept-2-ene; (3E)-1,1,1,2,2,3,4,5,6,6,7,7,7-tridecafluoro-5-methoxyhept-3-ene; (3E)-1,1,1,2,2,4,5,5,6,6,7,7,7-tridecafluoro-3-methoxyhept-3-ene

EC Number:

700-755-2

Molecular formula:

C8H3F13O

IUPAC Name:

(2E)-1,1,1,2,3,4,5,5,6,6,7,7,7-tridecafluoro-4-methoxyhept-2-ene; (3E)-1,1,1,2,2,3,4,5,6,6,7,7,7-tridecafluoro-5-methoxyhept-3-ene; (3E)-1,1,1,2,2,4,5,5,6,6,7,7,7-tridecafluoro-3-methoxyhept-3-ene

Details on test material:

- Purity: >99% (wt%)

Method

Target gene:

histidine (S. typhimurium), tryptophan (E. coli)

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:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Toxicity-mutation test: 33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 μg/plate

Mutagenicity test: 333, 667, 1000, 3333, and 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Based on the solubility of the test substance and compatibility with the target cells.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

ethanol

Negative solvent / vehicle controls:

no

True negative controls:

no

Controls 2

Positive controls:

other: 2-nitrofluorene (CAS 607-57-8; TA98 -S9); sodium azide (CAS 26628-22-8; TA100 and TA 1535 -S9); ICR-191 (CAS 17070-45-0; TA1537 -S9); 4-nitroquinoline-N-oxide (CAS 56-57-5; WP2uvrA -S9)

Positive control substance:

other: benzo(a)pyrene (CAS 50-32-8; TA98 +S9); 2-aminoanthracene (CAS 613-13-8; TA100, TA1535, TA1537, WP2uvrA +S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: In the non-activated assays, 0.5 mL of sham mix and 100 μL of vehicle, test substance dilution, or positive control were added to pre-heated (45–48°C) glass culture tubes containing 2 mL of selective top agar, followed by 100 μL of tester strain. In the S9-activated assays, 100 μL of the vehicle, test substance dilution, or positive control were added to pre-heated (45–48°C) glass culture tubes containing 2 mL of selective top agar, followed by 100 μL of tester strain and 0.5 mL of S9 mix. All mixtures were vortexed and overlaid onto the surface of minimum glucose agar plate. After the overlay solidified, the plates were inverted and incubated for approximately 49-69 hours at 37±2°C. Plates that were not evaluated immediately following incubation were stored at approximately 4°C

DURATION
- Preincubation period: none
- Exposure duration: approximately 49-69 hours at 37±2°C
- Expression time (cells in growth medium): approximately 49-69 hours

NUMBER OF REPLICATIONS: toxicity-mutation tests were plated in duplicate; mutagenicity tests plated in triplicate

NUMBER OF CELLS EVALUATED: 100 microliters of a tester strain culture that contained approximately 0.92E9 to 1.17E9 bacterial cells per milliliter were innoculated per plate

DETERMINATION OF CYTOTOXICITY: The appearance of the bacterial background lawn was assessed microscopically for test substance toxicity and precipitation. Toxicity was scored relative to the concurrent tester strain specific negative control, and evaluated as a decrease in the mean number of revertant bacterial colonies per plate. In addition, the thinning or disappearance of the bacterial background lawn was considered as signs of toxicity. A minimum of 3 non-toxic scorable dose levels were required to validate the study. A dose level was considered toxic if it caused a >50% reduction in the mean number of revertants per plate relative to the mean negative control value and exhibited a dose-dependent drop in the revertant count, or a reduction in the background lawn.

SCORING: Revertant colonies were counted with an automated colony counter. Plates that could not be accurately counted automatically were counted manually.

Evaluation criteria:

-Strains TA1535 and TA1537:
Data were judged positive if the increase in mean revertants at the highest numerical dose response was ≥ 3.0-fold the mean concurrent negative control value (vehicle control). This increase in the mean number of revertants per plate must be accompanied by a dose response associated with increasing concentrations of the test substance unless observed at the top dose level only.

-Strains TA98, TA100 and WP2uvrA:
Data sets were judged positive if the increase in mean revertants at the highest numerical dose response was ≥ 2.0-fold the mean concurrent negative control value (vehicle control). This increase in the mean number of revertants per plate must be accompanied by a dose response associated with increasing concentrations of the test substance unless observed at the top dose level only.

-Positive Control Values: Each mean positive control value must exhibit at least a 3.0-fold increase over the respective mean negative (vehicle) control value for each tester strain.

-Negative Control Values: The tester strain cultures must exhibit a characteristic mean number of spontaneous revertants per plate when plated along with the negative (vehicle) control under selective conditions. The acceptable ranges for the mean values of negative controls are: 8-60 for TA98, 60-240 for TA100, 4-45 for TA1535, 2-25 for TA1537, and 5-60 for WP2uvrA.

Statistics:

For each tester strain, the mean of the number of revertants and the standard deviations were calculated.

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

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

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

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

SOLUBILITY: The test substance was soluble in ethanol at 50 mg/mL, the highest stock concentration that was prepared for use on this study.

STERILITY CONTROLS: No contaminant colonies were observed on the sterility plates for the most concentrated test substance dilution (50 mg/mL) and the S9 and sham mixes.

RANGE-FINDING/SCREENING STUDIES: In the toxicity-mutation test, the maximum dose evaluated was 5000 μg/plate for tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA in the absence and presence of S9 metabolic activation. This dose was achieved using a concentration of 50 mg/mL and a 100 μL plating aliquot. The dose levels used in this test were 33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 μg/plate. The plate incorporation method was employed. This toxicity-mutation test was conducted 2 times. In the initial trial (T-1) the negative control values for TA98 with S9 activation were outside of the acceptable range. The data evaluated for this condition was collected in the second trial (T-2). No positive mutagenic responses were observed at any dose level in any tester strain in the absence or presence of S9 metabolic activation. No toxicity was observed at any dose level with any tester strain in either the absence or presence of S9. Test substance precipitation was observed starting at 3333 μg/plate in all test conditions except for TA98 both with and without S9 activation and WP2uvrA without S9 activation. For these test conditions precipitation was observed at 5000 μg/plate only.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Test substance precipitation was observed starting at 3333 μg/plate with all tester strains both with and without S9 activation.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this study, the test substance showed no evidence of mutagenicity in the Bacterial Reverse Mutation Test either in the presence or absence of Aroclor-induced rat liver S9. The test substance was concluded to be negative in this study.

This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).

Executive summary:

The test substance was evaluated for mutagenicity in the Bacterial Reverse Mutation Test using the plate incorporation method. Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2uvrA were tested in the absence and presence of an exogenous metabolic activation system (Aroclor-induced rat liver S9). The test was performed in 2 phases. The first phase was the toxicity-mutation test, which established the dose range for the mutagenicity test, and provided a preliminary mutagenicity evaluation. The second phase was the mutagenicity test, which evaluated and confirmed the mutagenic potential of the test substance. Ethanol was chosen as the dosing vehicle based on the solubility of the test substance and compatibility with the target cells. The test substance was soluble in ethanol at 50 mg/mL, the highest stock concentration that was prepared for use on this study.

In the toxicity-mutation test, the maximum dose evaluated was 5000 μg/plate for tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA in the absence and presence of S9 metabolic activation. This dose was achieved using a concentration of 50 mg/mL and a 100 μL plating aliquot. The dose levels used in this test were 33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 μg/plate. The plate incorporation method was employed. No positive mutagenic responses were observed at any dose level in any tester strain in the absence or presence of S9 metabolic activation. No toxicity was observed at any dose level with any tester strain in either the absence or presence of S9. Test substance precipitation was observed starting at 3333 μg/plate in all test conditions except for TA98 both with and without S9 activation and WP2uvrA without S9 activation. For these test conditions precipitation was observed at 5000 μg/plate only.

 

Based on the toxicity-mutation test, the maximum dose evaluated in the mutagenicity test was 5000 μg/plate for tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA in the absence and presence of S9 metabolic activation. This dose was achieved using a concentration of 50 mg/mL and a 100 μL plating aliquot. The dose levels used in this test were 333, 667, 1000, 3333, and 5000 μg/plate for all tester strains. The plate incorporation method was employed. No positive mutagenic responses were observed at any dose level or with any tester strain in either the absence or presence of S9 metabolic activation. No toxicity was observed at any dose level with any tester strain in either the absence or presence of S9. Test substance precipitation was observed starting at 3333 μg/plate with all tester strains both with and without S9 activation. All criteria for a valid study were met. Under the conditions of this study, the test substance showed no evidence of mutagenicity in the Bacterial Reverse Mutation Test either in the absence or presence of Aroclor-induced rat liver S9. It was concluded that the test substance was negative in this in vitro test.