2H-Pyran, 2-[4'-[difluoro(3,4,5-trifluorophenoxy)methyl]-3',5'-difluoro[1,1'-biphenyl]-4-yl]-5-ethyltetrahydro-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: In a bacterial reverse mutation test (plate incorporation test) according to OECD TG 471 the test item was not mutagenic in the absence and presence of a rat liver metabolizing system (S9 mix) (reference 7.6.1-1).

Chromosome Aberration test : In an in vitro chromosome aberration test according to OECD TG 473 the test item was not clastogenic (reference 7.6.1 -2).

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

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

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

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

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

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 05,2007 - May 14, 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: the V79 line was established from lung tissue of a male Chinese hamster by Ford and Yerganian in 1958
- Suitability of cells: cells were cloned to minimize the frequency of mutants, and to stabilize the karyotype

For cell lines:
- Absence of Mycoplasma contamination: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature:
DMEM (Dulbecco´s Minimal Essential Medium) supplemented with L-glutamine (4mM), sodium bicarbonate (0.375%), antibiotics (neomycine 0.015%) and 10% fetal calf serum
Cells were incubated at 37°C in 4-5% CO2 atmosphere (100% humidity)

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:

serie 1: 5.00, 15.8, 50.0, 158 and 500 µg/mL medium
serie 2: 50.0, 158 and 500 µg/mL medium

Vehicle / solvent:

Acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Griseofulvin

Remarks:

without S9 mix

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration duplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium
40-50 hours before treatment, an appropriate number of Quadriperm® dishes is prepared from a single pool of cells. Each part of a culture dish is seeded with 100 000 cells.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 5, 25 and 35 hours (without S9 mix) or 5 hours (with S9 mix)

FOR CHROMOSOME ABERRATION:
- Spindle inhibitor: colchicine, final concentration of 0.1 µg/mL, 3 hours incubation
- Methods of slide preparation and staining technique used including the stain used: The medium is replaced by KC1-solution (0.4 %) for hypotonic treatment. For fixation of cells fixative (methanol:acetic acid, 3:1) is added and then renewed 2 times. The slides are then stained in aceto-orcein, immersed in xylene and made permanent with Entellan.

- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): A total of 100 well spread metaphases are examined per culture (slide) at a magnification of 1250x, using an oil immersion phase contrast objective

- Determination of endoreplication and endoreplication: The frequency determination of polyploid cells and endoreduplications is based on scoring 1000 mitoses per slide, and estimation of the mitotic index on scoring 1000 cells per slide.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: MTT assay

Evaluation criteria:

After quality check of the cultures, code numbers (from a random list generated by an electronic program) were randomly assigned to the slides by a person not subsequently involved in slide evaluation. A total of 100 well spread metaphases are examined per culture (slide) at a magnification of 1250 x, using an oil immersion phase contrast objective.
Structural chromosome alterations are scored as follows:
Gap:
Achromatic region in chromatid(s) not greater than the width of a chromatid. Scored as gap (chromatid) or isogap (chromosomal).
Break:
Achromatic region in chromatid(s) greater than the width of a chromatid or a discontinuity with displacement. Scored as break (chromatid) or isobreak (chromosomal).
Exchange:
Aberrations arising from an exchange between one or two chromatids. These may be chromosome or chromatid interchanges. In studies of this type, where full karyotyping and chromosome banding are not performed, only asymmetrical or chromatid exchanges will normally be recognized.

Statistics:

Descriptive statistics
For all groups, mean values were calculated for the following parameters:
• the percentage of observed aberrant metaphases/culture (gaps included),
• the percentage of observed aberrant metaphases/culture (gaps excluded),
• the number of mitoses per 1000 cells/culture
• the number of polyploid cells per 1000 mitotic cells/culture

Statistical tests
For further statistical analysis the numbers of aberrant metaphases (gaps excluded) were used.
Pairwise comparisons within each experimental series. Each treatment group was compared with the negative control. For the comparisons the Exact Fisher Test was performed against one-sided alternatives.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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
- Data on pH: no change
- Data on osmolality: no change
- Precipitation and time of the determination: at >= 88.9 µg/mL
- Definition of acceptable cells for analysis: 100 wellspread metaphases per culture

RANGE-FINDING/SCREENING STUDIES: Due to limited solubility of the test item in solvents used for such in vitro tests, concentration ranging from 0.5 to 500 µg/mL. No clear cytotoxic effects were seen in the MMT assay.

STUDY RESULTS
- Results from cytotoxicity measurements: No clear cytotoxic effects, i.e. reduction in mitotic index or cell viability (MTT-test) were inducted by the test material.

- Genotoxicity results
The mean values of aberrant metaphases (gaps excluded) in the solvent control groups of the two experimental series ranged from 0.75 to 2.50 % and 1.50 to 2.50 % in the absence or presence of S9-mix, respectively. Thus the observed aberration rates were within the usual range of the experiments performed in our laboratory (cf. Historical data). A clear increase of aberrant metaphases was found for the positive controls, i.e. EMS and CPA. Statistical analysis in all cases yielded significant results. The experiments were therefore accepted as valid. For most of the test item treated cultures, the percentage of aberrant metaphases (gaps excluded) did not significantly exceed the percentages of the concurrent negative controls. One single value (1st experiment with S9 mix, 500 µg/mL) showed a weak statistical significance (*: 0.01> p > 0.05). However, this effect was not reproduced in the repeat experiment performed and, in addition, the value was not above the predefined historical negative control range (cf. Historical data). The effect seen in the first series was thus interpreted as a chance event of no biological relevance.
A clear increase was observed for the positive control for induction of polyploidy, i.e. griseofulvin. No treatment-related increase of polyploid cells was observed for the test item. No endoreduplications were observed.

HISTORICAL CONTROL DATA
- see attachments

Conclusions:

Treatment of V79 cell cultures with the test item, in the absence and presence of S9 mix, did not increase the proportion of cells with aberrant chromosomes. Furthermore,the test item did not increase the number of polyploid cells or endoreduplications under any experimental condition. Thus,the test item was not clastogenic in this in vitro test system.

Executive summary:

The test item was investigated in two experimental series for induction of chromosomal aberrations in V79 Chinese hamster cells in vitro. This also included examinations on whether or not the test material may have the potential to induce numerical aberrations, i.e. an increase in polyploidy. The following experimental conditions were selected in the absence and presence of an exogenous metabolizing system (S9 mix from livers of rats pretreated with Aroclor 1254): In the first series the cells have been exposed to the following concentrations in the presence and absence of metabolic activation: 5.00, 15.8, 50.0, 158 and 500 µg/mL.

In the second series the cells have been exposed to 50.0, 158 and 500 µg/mL.

The exposure times were the following:

-S9 mix: 5,25, and 35 hours

+S9 mix: 5 hours

After treatment of the cultures with the test material and the negative (solvent acetone) and positive controls for 5 h, the cultures are washed with Ca/Mg-free PBS and incubated in normal culture medium (DMEM) again. 22 hours after start of the treatment, colchicine is added (to yield a final concentration of 0.1 µg/mL) and the cultures are incubated for another 3 hours. Thus, the respective preparation time is 25 hours after start of the treatment with the test material.In the second experimental series in the absence of S9 mix, cells are continuously exposed to the test material, i.e. for 25 or 35 hours.

As positive control in the absence of S9 mix Ethylmethansulfonat and Griseofulvin were used. In the presence of S9 mix Cyclophosphamide was used as positive control.

The positive control test materials induced the expected clear increase in the proportion of cells with chromosomal aberrations. Griseofulvin induced a clear increase in the number of polyploid cells. the test item precipitated in the culture medium at concentrations 88.9 µg/mL. No clear cytotoxic effects, i.e. reduction in mitotic index or cell viability (MTT-test), were induced by the test material. The mean values of aberrant metaphases (gaps excluded) in the negative controls of both experiments (- and + S9 mix) ranged from 0.75 % to 2.50 %. test item did not show any relevant increase in the number of aberrant metaphases. Furthermore, no treatment-related increase of endoreduplications or polyploid cells was observed. I.e. neither structural nor numerical aberrations were detected.

In conclusion, treatment of V79 cell cultures with the test item, did not increase the proportion of cells with aberrant chromosomes. the test item was not clastogenic in this in vitro test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test

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 (reference 7.6.1 -1).

Chromosome Aberration Test

The test item was investigated in two experimental series for induction of chromosomal aberrations in V79 Chinese hamster cells in vitro. This also included examinations on whether or not the test material may have the potential to induce numerical aberrations, i.e. an increase in polyploidy. The following experimental conditions were selected in the absence and presence of an exogenous metabolizing system (S9 mix from livers of rats pretreated with Aroclor 1254):In the first series the cells have been exposed to the following concentrations in the presence and absence of metabolic activation: 5.00, 15.8, 50.0, 158 and 500 µg/mL.

In the second series the cells have been exposed to 50.0, 158 and 500 µg/mL.

The exposure times were the following:

-S9 mix: 5,25, and 35 hours

+S9 mix: 5 hours

After treatment of the cultures with the test material and the negative (solvent acetone) and positive controls for 5 h, the cultures are washed with Ca/Mg-free PBS and incubated in normal culture medium (DMEM) again. 22 hours after start of the treatment, colchicine is added (to yield a final concentration of 0.1 µg/mL) and the cultures are incubated for another 3 hours. Thus, the respective preparation time is 25 hours after start of the treatment with the test material.In the second experimental series in the absence of S9 mix, cells are continuously exposed to the test material, i.e. for 25 or 35 hours.

As positive control in the absence of S9 mix Ethylmethansulfonat and Griseofulvin were used. In the presence of S9 mix Cyclophosphamide was used as positive control.

The positive control test materials induced the expected clear increase in the proportion of cells with chromosomal aberrations. Griseofulvin induced a clear increase in the number of polyploid cells. the test item precipitated in the culture medium at concentrations 88.9 µg/mL. No clear cytotoxic effects, i.e. reduction in mitotic index or cell viability (MTT-test), were induced by the test material. The mean values of aberrant metaphases (gaps excluded) in the negative controls of both experiments (- and + S9 mix) ranged from 0.75 % to 2.50 %. test item did not show any relevant increase in the number of aberrant metaphases. Furthermore, no treatment-related increase of endoreduplications or polyploid cells was observed. I.e. neither structural nor numerical aberrations were detected.

In conclusion, treatment of V79 cell cultures with the test item did not increase the proportion of cells with aberrant chromosomes. the test item was not clastogenic in this in vitro test system (reference 7.6.1 -2).

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered not to be classified for genotoxicity under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/521.