Trans-2-{4-[difluoro(3,4,5-trifluorophenoxy)methyl]-3,5-difluorophenyl}-5-(trans-4-propylcyclohexyl)-1,3-dioxane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay (Ames): negative; reference 7.6.1 -1

Chromosome aberration test in vitro: negative; 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:

2011-09-13 to 2011-11-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

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

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 / 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 / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

his D 3052, uvrB, rfa + R-factor

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

his G 46, uvrB, rfa + R-factor

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

his G 428, rfa + R-factor

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

E. coli WP2

Details on mammalian cell type (if applicable):

his C 3076, uvrB, rfa

Additional strain / cell type characteristics:

other: mutations in the tryptophan operon

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate (S9 mix) with standard co-factors with metabolic activation (Aroclor)

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: 1.58, 5.00, 15.8, 50.0, 158, 500 and 1580 µg/plate
2. Series: 1.58, 2.81, 5.00, 15.8, 50.0 and 158 µg/plate

Vehicle / solvent:

Acetone

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

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:

The assessment of test item-induced effects is dependent on the number of spontaneous revertants of each bacterial strain (solvent controls) and the increase in the number of revertants at the test item concentration which shows the highest number of colonies. The following criteria, based upon the historical controls of the laboratory and statistical considerations, are established:

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

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

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

Rationale for test conditions:

According to the guideline.

Evaluation criteria:

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 item 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 and the results of each series scould be discussed case by case.

Statistics:

not required according to OECD TG 471

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

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, but tested up to precipitating concentrations

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, but tested up to precipitating concentrations

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, but tested up to precipitating concentrations

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, but tested up to precipitating concentrations

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Please refer to the attached background document.

Conclusions:

With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.

Executive summary:

The objective of this study was to provide information on possible health hazards for the test item and serve as a rational basis for risk assessment to the genotoxic potential of the test item in man.

The 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 coil 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 with TA 98, TA 100, TA 102,TA 1535 and TA 1537, and Escherichia coil WP2 uvr. In the two series with S9 mix, 10 or 30 % S9 in the S9 mix were used in the 1st and 2nd series, respectively. The test item was dissolved in acetone and tested at concentrations ranging from 1.58 to 1580 µg/plate.

Precipitation of the test item 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 a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. In both series, each performed with and without the addition of rat liver S9 mix as the external metabolizing system, the test item showed no increase in the number of revertants of the bacterial strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coil WP2 uvr. According to the criteria for negative and positive results predetermined in the Study Protocol, the test item was not mutagenic under the experimental conditions described. With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic 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:

2011-10-21 to 2012-04-19

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:

adopted 21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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
- Source of cells:Hoffmann-La Roche, Pharma, Basel, at May 27, 1997
The cells have a spontaneous aberration rate of about 0-5 % aberrant metaphases.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The cells were cultured in DMEM supplemented with L-glutamine (4 mM), sodium bicarbonate (0.375 %), antibiotics (neomycine 0.015 %), and 10 % fetal calf serum (FCS). All incubations were performed at +37°C in a 4-5 % carbon dioxide atmosphere (100 % humidity). Before use, serum batches were screened for efffects on cloning efficiency and cell growth.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix with S9 from Aroclor 1254 induced male rats

Test concentrations with justification for top dose:

Solubility experiments performed prior to the study start revealed the the test item was soluble in acetone at concenttrations <158 g/mL. Hence, the top concentration tested was limited by solubility.
The test item precipitated in the culture medium at concenrations above 88.9 µg/mL in the presence of S9 mix only (5 hours exposure time).
A change in the pH or the osmotic value of the cell culture medium did not occur in the dose range tested.
No clear cytotoxic effects, i.e. reduction in cell viability (MTT test) or mitotic index below 50% were induced by the test item.
For these reasons, cultures treated with the following test material concentrations were evaluated in absence and presence of S9 mix for the occurrence of chromosomal aberrations:
Experiment 1 (with and without S9 mix): 15.8, 50.0 and 158 µg/mL
Experiment 2 (without S9 mix): 15.8, 50.0 and 158 µg/mL

Vehicle / solvent:

Acetone, 0.1% in the culture medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: Griseofulvin

Remarks:

- S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

+ S9 mix

Details on test system and experimental conditions:

A total of 100 metaphases were examined per culture for cytogenetic damage. Additionally, a total of 500 metaphases was examined per culture for the occurence of polyploidies.

Exposure times: -S9 mix: 5, 25 and 31 hours
+S9 mix. 5 hours

Solvent for the test material: Acetone

Positive controls: - S9 mix: 250 or 500 µg Ethylmethansulfonat (EMS)/mL
31.6 µg Griseofulvin (GRIS)/mL

+S9 mix: 2.00 µg Cyclophosphamide (CPA)/mL

Evaluation criteria:

The decisive parameter for the evaluation of both, the treated and untreated cultures, is the number of aberrant metaphases (excluding gaps) per 100 cells. A basic prerequisite for the acceptance of a test series is (a) the correspondence of the actual negative controls with the historic negative controls of the laboratory and (b) a statistically significant and biologically relevant increase in the number of aberrant metaphases for the respective positive controls in relation to the actual negative controls. The discussion of biological relevance includes, among other things, considerations concerning the type and time dependent appearance of the observed chromosomal aberrations.
A test material is defined as being unambiguously negative or non-clastogenic in this test system if no statistically significant increase in the number of aberrant metaphases per 100 cells, as compared to the actual negative control, occurs at any test material concentration.
A test material is positive or clastogenic in this test system if
• a statistically significant, dose-related increase in the number of aberrant metaphases per 100 cells occurs or
• a statistically significant increase in the number of aberrant metaphases per 100 cells is reproduced at the same test material concentration in independent experiments.
In both cases, however, the number of aberrant metaphases should be above the range defined as the historical negative controls of the laboratory and the biological relevance of the results has to be discussed.
In all other cases, further decisions for testing strategies should be made following the scientific evaluation of all existing data including those of non­ toxicological investigations.

Statistics:

Fisher’s Exact Test

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 applicable

True negative controls validity:

not examined

Positive controls validity:

valid

Please refer to the attached background material.

Conclusions:

In conclusion, treatment of V79 cell cultures with the test item did not increase the proportion of cells with aberrant chromosomes in two independent experimental series in absence and presence of metabolic activation (S9 mix). The test material was not clastogenic in this in vitro test system.

Executive summary:

The test material 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 endoreduplication or 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):

No. of slides per concentration:

Solvent control:               4

Others:                            2

No. of metaphases evaluated per slide: 100 (for structural aberrations); 500 (for polyploidy)

Preparation times: 25 and 35 hours

Exposure times:       -S9 mix: 25, and 31 hours

+S9 mix: 5 hours

Solvent for the test material: Acetone

Concentrations evaluated (+/- S9 mix):

Experiment 1: 15.8, 50.0 and 158 µg/mL

Experiment 2: 15.8, 50.0 and 158 µg/mL

Positive controls:        - S9 mix:             500 / 250 µg Ethylmethansulfonat (EMS)/mL (1st / 2nd experiment)

                                                            31.6 µg Griseofulvin (GRIS)/mL

                                   +S9 mix:            2.00 µg Cyclophosphamide (CPA)/mL

The mean values of aberrant metaphases (gaps excluded) in the negative controls of both experiments (in the absence and presence of S9 mix) ranged from 1.3 to 3.3 % and thus met the historical negative control range. The positive control test materials, EMS and CPA, induced the expected clear increase in the proportion of cells with chromosomal aberrations. Griseofulvin induced a clear increase in the number polyploid cells. Precipitation of the test item in the culture medium was only seen in the experimental part in the absence of S9 mix at concentraions > 88.9 µg/mL. No clear cytotoxic effects, i.e. reduction in mitotic index or cell viability (MTT-test) were induced by the test item. The limiting factor for the highest test concentration was the solubility of the test item. Treatment of V79 cells with the test item did not lead to a biologically relevant increase in the proportion of cells with aberrant chromosomes. Furthermore, no relevant treatment-related increase in endomitotic metaphases or polyploid cells was observed.

In conclusion, treatment of V79 cell cultures with the test item did not relevantly increase the proportion of cells with aberrant chromosomes in two independent experimental series in absence and presence of metabolic activation (S9 mix). 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

Bacterial reverse mutation assay, reference 7.6.1 -1

The objective of this study was to provide information on possible health hazards for the test item and serve as a rational basis for risk assessment to the genotoxic potential of the test item in man. The 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 coil 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 with TA 98, TA 100, TA 102,TA 1535 and TA 1537, and Escherichia coil WP2 uvr. In the two series with S9 mix, 10 or 30 % S9 in the S9 mix were used in the 1st and 2nd series, respectively. The test item was dissolved in acetone and tested at concentrations ranging from 1.58 to 1580 µg/plate. Precipitation of the test item 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 a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. In both series, each performed with and without the addition of rat liver S9 mix as the external metabolizing system, the test item showed no increase in the number of revertants of the bacterial strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coil WP2 uvr. According to the criteria for negative and positive results predetermined in the Study Protocol, the test item was not mutagenic under the experimental conditions described. With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.

Chromosome Aberration assay in vitro, reference 7.6.1 -2

The test material 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 endoreduplication or 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):

No. of slides per concentration:

Solvent control:               4

Others:                            2

No. of metaphases evaluated per slide: 100 (for structural aberrations); 500 (for polyploidy)

Preparation times: 25 and 35 hours

Exposure times:       -S9 mix: 25, and 31 hours

+S9 mix: 5 hours

Solvent for the test material: Acetone

Concentrations evaluated (+/- S9 mix):

Experiment 1: 15.8, 50.0 and 158 µg/mL

Experiment 2: 15.8, 50.0 and 158 µg/mL

Positive controls:      - S9 mix:             500 / 250 µg Ethylmethansulfonat (EMS)/mL (1st / 2nd experiment)

                                                                31.6 µg Griseofulvin (GRIS)/mL

                                   +S9 mix:            2.00 µg Cyclophosphamide (CPA)/mL

The mean values of aberrant metaphases (gaps excluded) in the negative controls of both experiments (in the absence and presence of S9 mix) ranged from 1.3 to 3.3 % and thus met the historical negative control range. The positive control test materials, EMS and CPA, induced the expected clear increase in the proportion of cells with chromosomal aberrations. Griseofulvin induced a clear increase in the number polyploid cells. Precipitation of the test item in the culture medium was only seen in the experimental part in the absence of S9 mix at concentraions > 88.9 µg/mL. No clear cytotoxic effects, i.e. reduction in mitotic index or cell viability (MTT-test) were induced by the test item. The limiting factor for the highest test concentration was the solubility of the test item. Treatment of V79 cells with the test item did not lead to a biologically relevant increase in the proportion of cells with aberrant chromosomes. Furthermore, no relevant treatment-related increase in endomitotic metaphases or polyploid cells was observed.

In conclusion, treatment of V79 cell cultures with the test item did not relevantly increase the proportion of cells with aberrant chromosomes in two independent experimental series in absence and presence of metabolic activation (S9 mix). The test item was not clastogenic in this in vitro test system.

Justification for classification or non-classification

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

The available experimental test data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance does not require classification as genotoxic under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/521.