trans-2-[4'-[difluoro(3,4,5-trifluorophenoxy)methyl]-2,3',5'-trifluoro[1,1'-biphenyl]-4-yl]-5-butyl-1,3-dioxane

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

Study period:

27.11-06.12.2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

8 June 2000

Deviations:

no

Principles of method if other than guideline:

n.a.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

trans-2-[4'-[difluoro(3,4,5-trifluorophenoxy)methyl]-2,3',5'-trifluoro[1,1'-biphenyl]-4-yl]-5-butyl-1,3-dioxane

EC Number:

695-906-1

Cas Number:

1256753-06-6

Molecular formula:

C27H22F8O3

IUPAC Name:

trans-2-[4'-[difluoro(3,4,5-trifluorophenoxy)methyl]-2,3',5'-trifluoro[1,1'-biphenyl]-4-yl]-5-butyl-1,3-dioxane

Test material form:

solid

Method

Target gene:

HIS operon (S. thyphimurium)
TRP operon (E. coli)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

his D 3052, uvrB-, rfa- + R-factor (pKM101)

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

his G 46, uvrB-, rfa- + R-factor (pKM101)

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

his G 46, uvrB-, rfa-

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

his C 3076, uvrB-, rfa-

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain 5

Species / strain / cell type:

E. coli WP2

Details on mammalian cell type (if applicable):

trp-, uvrA-

Additional strain / cell type characteristics:

other: mutations in the tryptophan operon

Species / strain 6

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

his G428, rfa + R-factor

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from Aroclor1254-pretreated rats with standard co-factors

Test concentrations with justification for top dose:

The test material concentrations used were selected according to the EC, OECD and Japanese guidelines for this test system. The test material showed best solubility in Acetone and 5000 µg/plate was chosen as the appropriate maximum concentration.
1. Series: 5.00, 15.8, 50.0, 158, 500, 1580 and 5000 µg/plate
2. Series: 5, 15.8, 28.1, 50, 88.9 and 158 µg/plate

Vehicle / solvent:

Acetone

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

cumene hydroperoxide

other: Daunomycin

Remarks:

without S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

with S9

Details on test system and experimental conditions:

Bacterial strains were tested in accordance with the plate incorporation method. 3 parallel plates were used for each concentration step of the test material. The incubation of plates was performed at 36-38°C for 2 days. Liver S9 mix from rats pre-treated with Aroclor 1254 was used as the metabolic activation system. Two experimental series were performed, containing 10% S9 ind the 1st and 30% S9 in the 2nd series.

Rationale for test conditions:

according to Guideline

Evaluation criteria:

A test material was to be defined as positive or mutagenic in this assay if
• the assay is considered valid and
• a biologically relevant increase in the mean number of revertants above a threshold of 2-fold (TA 98, TA 100, WP2 uvrA) or 3-fold (TA 1535, TA 1537) as compared to the con-current negative controls is observed
• an increase exceeding the threshold at only one concentration is considered as biologically meaningful if reproduced in a second independent experiment
• a concentration-dependent increase is considered biologically meaningful if the threshold is exceeded at more than one concentration

A test material is defined as negative or non-mutagenic in this assay if
• the assay is considered valid and
• none of the above-mentioned criteria are met

Whenever colony counts remain within the historical range of negative controls, such increases are considered as biologically not meaningful. In general, two series of experiments must be performed. However, there is no requirement for verification of a clear positive response (OECD TG 471, 1997).

Results which only partially satisfied the above criteria were dealt with on a case by case basis. Biological relevance was considered, for example consistency of response within and between concentrations and (where applicable) between experiments.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

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:

cytotoxicity

Remarks:

>=500 µg/plate

Vehicle controls validity:

valid

Untreated 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:

cytotoxicity

Remarks:

>=1580 µg/plate

Vehicle controls validity:

valid

Untreated 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:

cytotoxicity

Remarks:

>=1580 µg/plate

Vehicle controls validity:

valid

Untreated 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:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=889 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=889 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- Precipitation: >=158 µg/plate

Applicant's summary and conclusion

Conclusions:

In both series of experiments, each performed with and without the addition of rat liver S9 mix
as the extemal metabolizing system, the test item showed no increase in the number of revertants
of any bacterial strain. According to the criteria for negative and positive results, the test item was not mutagenic under
the described experimental conditions.

Executive summary:

Objective

The present study was conducted to investigate the test material for mutagenic potential in a bacterial reverse gene mutation assay in the absence and presence of a rat liver metabolizing system (S9 mix).

Study Design

The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA102, TA 1535 and TA 1537, and Escherichia coil WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from rats pre-treated with Aroclor 1254 was used. In this study, two independent experimental series were performed. The S9 mix used contained 10% S9 in the 1st and 30% S9 in the 2nd series. The test item was dissolved in Acetone and tested at concentrations ranging from 5 to 5000 µg/plate.

Results

Precipitation of the test material on the agar plates occurred at concentrations >=158.

The mean numbers of revertant colonies were all within acceptable ranges for solvent control treatments, or were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

Under the conditions described, the test item induced no increases in revertant numbers.

Conclusion

Based on these results for the described experimental conditions, it was concluded that the test item was not mutagenic in this bacterial reverse mutation test .