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Toxicological information

Genetic toxicity: in vitro

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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:
other: GLP Guideline Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Liquid
Details on test material:
Name : C4 OLEFIN (PFBE)
Chemical name : 3,3,4,4,5,5,6,6,6-nonafluoro-1-hexene
Purity : 100%
CAS Reg. number 1 : 19430-93-4
Appearance : clear, colourless liquid
Batch number : 91206
Molecular Formula : C6H3F9
Molecular weight : 246 g/mol
Boiling point : 60 °C
Storage conditions : 2-10 °C, protected from light
Date of receipt : 22 February 2010
Expiry date : 31 March 2011
Supplier : Sponsor
TNO dispense reference no. : 100039

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 rat liver homogenate
Test concentrations with justification for top dose:
0.062, 0.185, 0.556, 1.667, 5.0 ul/plate
Vehicle / solvent:
ethanol
Controls
Untreated negative controls:
yes
Remarks:
ethanol
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-aminoanthracene, N-ethyl-N-nitrosourea
Details on test system and experimental conditions:
Fresh bacterial cultures were prepared by inoculating nutrient broth with a thawed aliquot of the stock culture and incubating the broth for approximately 10-16 h at ca. 37 oC while shaking. For each test concentration, 25 µl of the test substance solution or negative control, 0.1 ml of the bacterial suspension and 0.575 ml of the S9-mix with metabolic activation or 0.575 ml sodium phosphate 100 mM (pH 7.4) without metabolic activation were pre-incubated for 3.0 hours at 37.1ºC. Bacterial suspensions were stirred during pre-incubation.
To 2 ml molten top agar (containing 0.6 % agar, 0.5 % NaCl and 0.05 mM L-histidine.HCl/0.05 mM biotin or 0.05 mM tryptophane for the S. typhimurium strains, and E. coli WP2 uvrA strain, respectively), maintained at ca. 46 degC, was added 0.7 ml of the pre-incubation mixture of the appropriate strain. For the positive control 0.1 ml of the positive control substance solution, and 0.5 ml S9-mix for the experiments with metabolic activation or 0.5 ml sodium phosphate 100 mM (pH 7.4) for the experiments without metabolic activation were added to 2 ml molten top agar. The ingredients were thoroughly mixed and the mix was immediately poured onto minimal glucose agar plates (1.5 % agar in Vogel and Bonner medium E with 2 % glucose). All determinations were made in triplicate. The plates were incubated at ca. 37 degC for approximately 76 hours. Subsequently, the his+ and trp+ revertants were counted.
Evaluation criteria:
The mutagenicity study is considered valid if the mean colony counts of the vehicle control values of the strains are within acceptable ranges, if the results of the positive controls meet the criteria for a positive response, if no more than 5 % of the plates are lost through contamination or other unforeseen events and if at least 3 doses are non toxic.

A test substance is considered to be positive in the bacterial gene mutation test if the mean number of revertant colonies on the test plates increased in a concentration- related manner or if a two-fold or greater increase is observed compared to the negative control plates. A clear positive response does not need to be verified. Marginally or weakly positive results should be verified by additional testing.

A test substance is considered to be negative in the bacterial gene mutation test if it produces neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points.

Positive results from the bacterial reverse mutation test indicate that a substance induces point mutations by base pair substitutions or frameshifts in the genome of either Salmonella typhimurium and/or Escherichia coli. Negative results indicate that under the test conditions, the test substance is not mutagenic in the tested strains.

Omission of a second test under these conditions is acceptable as a single test does not, or hardly ever results in false negative conclusions (TNO historical data and Kirkland and Dean, 1994).

Both numerical significance and biological relevance are considered together in the evaluation. No statistical analysis was performed.
Statistics:
No statistical analysis was performed.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

One bacterial reverse mutation test was performed. Ethanol was used as vehicle; a clear colourless solution was obtained. Five concentrations were tested, ranging from 0.062 to 5.0 µl per plate. Negative controls (ethanol) and positive controls were run simultaneously with the test substance.

In the presence of S9-mix, in strain TA1537 the negative control was slightly outside the acceptable range (45 revertants, range 4-40 revertants). Since no increase or decrease in the mean number of revertants was observed at any concentration of the test substance, this slight increase in the negative control was considered an artifact and not biologically relevant.

The mean numbers of his+ and trp+ revertant colonies of the negative controls in strains TA 1535, TA 98, TA 100 and WP2 uvrA were within the acceptable range and in all strains the positive controls gave the expected increase in the mean numbers of revertant colonies. The test was regarded as valid.

The test substance was not toxic to any strain, in both the absence and presence of S9- mix, as neither a decrease in the mean number of revertants nor a clearing of the background lawn of bacterial growth compared to the negative controls was observed.

In both the absence and presence of S9-mix in strains TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA, C4 Olefin did not induce a minimal 2-fold and/or dose related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that the results obtained with the test substance in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA, in both the absence and the presence of the S9-mix, indicate that PFBE (3,3,4,4,5,5,6,6,6-nonafluorohexene) is not mutagenic under the conditions employed in this study.
Executive summary:

PFBE (3,3,4,4,5,5,6,6,6-nonafluorohexene) was examined for mutagenic activity in the bacterial reverse mutation test using the histidine-requiring Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the tryptophan- requiring Escherichia coli strain WP2 uvrA, in the absence and presence of a liver fraction of Aroclor 1254-induced rats for metabolic activation (S9-mix).

The test substance was diluted in ethanol. One bacterial reverse mutation test was performed. All strains were used, in the absence and presence of S9-mix, with five concentrations of the test substance, ranging from 0.062 to 5.0 µl/plate. Negative controls (ethanol) and positive controls were run simultaneously with the test substance.

In the presence of S9-mix, in strain TA1537 the negative control was slightly outside the acceptable range (45 revertants, range 4-40 revertants). Since no increase or decrease in the mean number of revertants was observed at any concentration of the test substance, this slight increase in the negative control was considered an artifact and not biologically relevant.

The mean numbers of his+ and trp+ revertant colonies of the negative controls in strains TA 1535, TA 98, TA 100 and WP2 uvrA were within the acceptable range and in all strains the positive controls gave the expected increase in the mean numbers of revertant colonies. The test was regarded as valid.

PFBE was not toxic to any strain, in both the absence and presence of S9-mix, as neither a decrease in the mean number of revertants nor a clearing of the background lawn of bacterial growth compared to the negative controls was observed.

In both the absence and presence of S9-mix in all strains, PFBE did not induce a more than 2-fold and/or dose related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control.

It is concluded that the results obtained with the test substance in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA, in both the absence and the presence of the S9-mix, indicate that PFBE (3,3,4,4,5,5,6,6,6-nonafluorohexene) is not mutagenic under the conditions employed in this study.