3,3,4,4,4-pentafluorobut-1-ene

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:

23rd May - 9th June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

The unplanned deviations are thought not to affect the validity, integrity or the result of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The unplanned deviations are thought not to affect the validity, integrity or the result of the study.

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The unplanned deviations are thought not to affect the validity, integrity or the result of the study.

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries

Deviations:

yes

Remarks:

The unplanned deviations are thought not to affect the validity, integrity or the result of the study.

Principles of method if other than guideline:

The purpose of the study was to evaluate the ability of the test substance, 3, 3, 4, 4, 4-pentafluorobut-1-ene to induce reverse mutations, either directly or after metabolic activation, at the histidine or tryptophan locus in the genome of five strains of bacteria.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3,3,4,4,4-pentafluorobut-1-ene

EC Number:

206-775-3

EC Name:

3,3,4,4,4-pentafluorobut-1-ene

Cas Number:

374-27-6

Molecular formula:

C4H3F5

IUPAC Name:

3,3,4,4,4-pentafluorobut-1-ene

Test material form:

gas

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Identification: 3, 3, 4, 4, 4-pentafluorobut-1-ene
- Physical state/appearance: colourless gas
- Batch No. of test material: 3 1101
- Expiration date of the lot/batch: 26 November 2017
- Purity: 99.753%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark.
- Stability under test conditions: all formulations were used within 4 hours of preparation and were assumed to be stable for this period.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: atmospheres of the test substance were generated by mixing the gas under test with clean dry air.

Method

Target gene:

Histidine or tryptophan locus.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: University of California, Berkley, on culture dishes (04 August 1995); British Industrial Biological Research Association, on a nutrient agar plate (17 August 1987)
- Suitability of cells:
- Cell cycle length, doubling time or proliferation index:
- Number of passages if applicable:
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes:
- Normal (negative control) cell cycle time:

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: top agar prepared using 0.6% Bacto agar and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates weer purchased for the preliminary toxicity test, Experiment 1 and 2.
- Properly maintained: yes
- Periodically checked: yes - each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Test concentrations with justification for top dose:

Preliminary test: 3.125, 6.25, 12.5, 25 and 50%.
Experiment 1: 3.125, 6.25, 12.5, 25 and 50%. The maximum recommended dose level was 50%.
Experiment 2: 3.125, 6.25, 12.5, 25 and 50%.

Vehicle / solvent:

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

Controls

Untreated negative controls:

yes

Remarks:

Untreated (to assess the spontaneous revertant colony rate)

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle control = air

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: overnight sub-cultures of the stock cultures were prepared in nutrient broth and incubated at 37°C for approximately 10 hours.
- Exposure duration: approximately 48 hours at 37 ± 3°C

NUMBER OF CELLS EVALUATED:
- The presence of revertant colonies were scored using an automated colony counting system.

DETERMINATION OF CYTOTOXICITY
- Method: plates were viewed microscopically for evidence of thinning (toxicity)

Evaluation criteria:

There are several criteria for determining a positive result. Any one or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested.
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response)

A test substance is considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments give clear positive or negative results, in some instances, the data generated will prohibit making a definite judgement about test substance activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test results

Key result

Species / strain:

other: S.typhimurium TA1535, TA98, TA1537, TA100, E.coli WP2uvrA

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

Additional information on results:

Cytotoxicity - several manual counts were required due to revertant colonies spreading slightly and on the edges of the plates, thus distorting the actual plate count.

Any other information on results incl. tables

Preliminary toxicity test

- The test substance was non-toxic to the strains of bacteria used (TA100 and WP2uvrA).

- Salmonella strain TA100 (absence of S9 -mix) plate counts were slightly below expected levels, but were still considered valid, since the test was performed only to assess test substance toxicity in order to evaluate dose levels for the main phase of testing.

Mutation test

- The characteristics, viability and spontaneous reversion rate of the master strains were found to be satisfactory and the amino acid supplemented top agar and the S9 -mix used in both experiments were shown to be sterile.

- The spontaneous mutation rates for the negative (untreated) controls were considered to be acceptable.

- The maximum dose level of the test substance in the first experiment was selected as the maximum recommended dose level of 50% in air.

- There was no visible reduction in the growth of the bacterial background lawn at any test substance concentration, either in the presence of absence of metabolic activation (S9 -mix) in the first or second mutation test and no visible reduction in the presence of absence of the S9 -mix at any test concentration in the second mutation test.

- There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test substance concentration, either with or without metabolic activation in Experiment 1 or 2.

- A small statistically significant increase in revertant colony frequency was observed in the first mutation test at 25% per plate (TA1537 in the presence of the S9 -mix), but it was considered to be of no biological relevance as there was no evidence of a dose-response relationship or reproducibility.

- Individual revertant counts at the statistically significant dose level was within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was 1.9 times the concurrent vehicle controls.

- The vehicle (air) control plates gave counts of revertant colonies within the normal range.

- All of the positive control chemicals used in the test, induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Therefore, the sensitivity of the assay and efficacy of the S9 -mix were validated.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the test substance, 3,3,4,4,4-pentafluorobut-1-ene (CAS No 374-27-6) was considered to be non-mutagenic in Salmonella typhimurium strains (TA1537, TA98, TA1535, TA100) and the Escherichia coli WP2uvrA strain.

Executive summary:

The aim of this study was to evaluate the ability of the test substance, 3, 3, 4, 4, 4 -pentafluorobut-1 -ene, to induce reverse mutations, either directly or after metabolic activation, at the histidine or tryptophan locus in the genome of five strains of bacteria (Salmonella typhimurium TA1537, TA98, TA1535 and TA100, and Escherichia coli WP2uvrA). The test method conducted was designed to be compatible with the following guidelines: OECD 471, EU Method B13/14, EPA OPPTS 870 .5100 and the Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.

The five bacterial strains were tested again atmospheres of the test substance using the Ames bacterial/microsome test system, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 3.125 -50% (air mixtures) in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material atmospheres.

A preliminary toxicity test was conducted in order to select appropriate dose levels for the main phase of testing and evaluate test substance toxicity. The test substance was shown to be non-toxic to the bacterial strains used (TA100 and WP2uvrA). TA100 strain plate counts in the absence of the S9 -mix were slightly below expected levels, however the results were still considered valid. The test concentrations chosen were 3.125, 6.25, 12.5, 25 and 50%, which were employed for both Experiment 1 and 2.

The maximum dose level of the test substance in the first experiment was selected as the maximum recommended dose level of 50% in air. There was no visible reduction in the growth of the bacterial background lawn at any test substance concentration, either in the presence or absence of metabolic activation (S9 -mix) in the first mutation test, and the same maximum dose level was used in the second mutation test. There was also no visible reduction in the growth of the bacterial background lawn at any test substance concentration with or without the S9 -mix in the second mutation test. There were no significant increases in the frequency of revertant colonies seen for any of the bacterial strains with any dose of test material with or without metabolic activation in Experiment 1 or 2, except a small statistically significant increase in revertant colony frequency seen in the first mutation test at 25% per plate (TA1537 in the presence of S9 -mix). However, this increase was considered not to be biologically relevant, as there was no evidence of a dose-response relationship or reproducibility. Also, the individual revertant counts at the statistically significant dose level was within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.9 times the concurrent vehicle controls.

The vehicle (air control) gave counts of revertant colonies within the normal range and all of the positive control chemicals induced marked increases in the frequency of revertant colonies in both the presence and absence of metabolic activation. Therefore, the sensitivity of the assay and efficacy of the S9 -mix were validated.

Under the conditions of this study, the test substance, 3,3,4,4,4-pentafluorobut-1-ene (CAS No 374-27-6) was considered to be non-mutagenic in Salmonella typhimurium strains (TA1537, TA98, TA1535, TA100) and the Escherichia coli WP2uvrA strain.