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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006-01-10 to 2006-05-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2000/32/EC
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997-07-21
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-{4'-[difluoro(3,4,5-trifluorophenoxy)methyl]-3',5'-difluoro-[1,1'-biphenyl]-4-yl}-5-ethyloxane
EC Number:
616-651-4
Cas Number:
787582-75-6
Molecular formula:
C26H21F7O2
IUPAC Name:
2-{4'-[difluoro(3,4,5-trifluorophenoxy)methyl]-3',5'-difluoro-[1,1'-biphenyl]-4-yl}-5-ethyloxane

Method

Target gene:
HIS operon (S. thyphimurium)
TRY operon (E. coli)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 102
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Due to migration, the value was transferred to one of the current document's attachments
Test concentrations with justification for top dose:
The test material concentrations were used selected according to the EC and OECD guidelines for this test system and the requirements of the Labor Ministry of Japan:
1. Series: 5, 15.8, 50, 158, 500, 1580 and 5000 µg per plate
2. Series: 50, 88.9, 158, 281 and 500 µg per plate
Vehicle / solvent:
Acetone
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Daunomycin
Remarks:
without S9 mix, TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 mix, TA100, TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cumene hydroperoxide
Remarks:
without S9 mix, TA102
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix, TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 mix, WP2 uvrA
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
with S9 mix, TA98, TA100, TA1535, TA1537, WP2 uvrA
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
without S9 mix, TA102
Details on test system and experimental conditions:
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 2 to 3 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition; reduction in the number of spontaneous revertants

METHODS FOR MEASUREMENTS OF GENOTOXICIY: revertant colonies

Evaluation criteria:
All further results, ranging between "no" and "clear", are assessed as "weak in-creases".

Maximal Mean Number of Colonies over the Actual Solvent Control
(Test Material)
-----------------------------------------------------------------------------------------

<=9 >=30
<=19 >=40
<=29 >=80
<=49 >=120
<=79 >=200
No increase Clear increase

A test material is defined as non-mutagenic in this assay if "no" or "weak increases" occur in the first and second series of the main experiment.
("Weak increases" randomly occur due to experimental variation)

A test material is defined as mutagenic in this assay if:
- a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;

- "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.

In all further cases, a third test series with the bacterial strain in question should be performed.
If the criteria for a positive test result are not fulfilled in at least two out of the three series, the test material is defined as being non-mutagenic in this test system.
Statistics:
NA

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
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
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: at concentrations above 158 µg/plate

STUDY RESULTS
- Concurrent vehicle negative and positive control data : see attached results

Ames test:
- Signs of toxicity : no cytotoxicity

HISTORICAL CONTROL DATA :
-----------------------------------------------------------------------------------------
Mean Number of Colonies Maximal Mean Number of Colonies over the Actual
(Solvent Control) Solvent Control
(Test Material)
-----------------------------------------------------------------------------------------

<=10 <=9 >=30
<=30 <=19 >=40
<=80 <=29 >=80
<=200 <=49 >=120
<=500 <=79 >=200
Assessment No increase Clear increase

-----------------------------------------------------------------------------------------

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions described test material was not mutagenic with and without addition of S9 mix as the external metabolizing system.
Executive summary:

Investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. In the two series with S9 mix, 10 or 30 % S9 mix were used in the first and second series, respectively.

The test item was dissolved in acetone and tested at concentrations ranging from 5 to 5000 µg/plate. Precipitation of the test material on the agar plates occurred at concentrations >= 158 µg/plate. Toxicity to the bacteria was not observed. Daunomycin, sodium azide, 9-aminoacridine, 4-nitroquinolin-N-oxide and cumene hydroperoxide served as strain specific positive control test materials in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the test materials used as positive controls led to clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. The test material was not mutagenic with and without addition of S9 mix as the external metabolizing system under the experimental conditions described.