2,3-difluoro-1-methoxy-4-[(trans,trans)-4'-propyl[1,1'-bicyclohexyl]-4-yl]-benzene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

GLP OECD 471: negative

GLP OECD 473: negative

Link to relevant study records

Reference

Reference 1

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:

Feb 05, 2003 - Jul 21, 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella: histidine operon
E coli. tryptophane operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

- Type and identity of media:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

- Type and identity of media:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from Aroclor 1254 pretreated rats

Test concentrations with justification for top dose:

1st series: 0.5, 1.58, 5.00, 15.8, 50.0, 158, and 500 µg/plate
2nd series: 5.00, 8.89, 15.8, 50.0, 88.9 µg/plate

Vehicle / solvent:

acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: Sodium azide, 2-Aminoanthracene, Cumene hydroperoxide, 9-Aminoacridine, Daunomycin, 4-Nitroquinoline-N-oxide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 3-5 hours
- Exposure duration: about 2 days


SELECTION AGENT (mutation assays): Histidine, Tryptophane

NUMBER OF REPLICATIONS:
Negative controls 6
Test material 3
Positive controls 3


NUMBER OF CELLS EVALUATED: about 1e9


DETERMINATION OF CYTOTOXICITY
- Method: other: background cytotox

Rationale for test conditions:

According to guideline

Evaluation criteria:

- valid assay (cf. laboratory historical data)
- no or weak increase in revertant colonies = negative
- clear, dose dependent (over at least two concentrations), and reproducible increase in revertant colonies= positve

Statistics:

not applied

Key result

Species / strain:

other: all strains/cell types tested

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Water solubility: ok
- Precipitation:yes ( ≥ 50 µg/plate)
- Other confounding effects: no

Conclusions:

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

Executive summary:

The informtion for this endpoint study record was obtained from an experimental study. The OECD GLP criteria were met and the methods applied are fully compliant with OECD TG 471. With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 27 - November 26, 2012

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)

Deviations:

no

Principles of method if other than guideline:

none

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):

- Type and identity of media: Minimal Essential Medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (induction using Aroclor 1254)

Test concentrations with justification for top dose:

2.81, 8.89, and 28.1 µg / mL

Vehicle / solvent:

Name: Acetone, purity (GC)  99 %
Final concentration: 0.1 %
Article number: 1.00013
Batch: K410113113

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

other: griseofulvin

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5, 25, and 31 hours (-S9) and 5 hours (+S9)


STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Solvent control: 4; others: 2

NUMBER OF CELLS EVALUATED: 100 metaphases (structural abberations); 1000 metaphases (polyploidy)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other:

OTHER:

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 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. Confirmation of negative results is not considered necessary if these criteria are fulfilled.

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.

There is no requirement for verification of a clear positive response. In both cases, however, the number of aberrant metaphases has to 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.

Statistics:

Standard statistical methods have been applied for data processing.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: yes (≥15.8 µg/mL)
- Cytotoxicity: yes (≥15.8 µg/mL)


ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test material precipitated in the culture medium at concentrations ≥ 15.8 µg/mL.
Cytotoxic effects, i.e. reduction in mitotic index or cell viability (MTT test), were induced by the test material at concentrations ≥ 15.8 µg/mL.
Thus, the limiting factors for the highest test concentration were cytotoxicity and solubility.

see attachement

Conclusions:

The treatment of V79 cell cultures with the test material did not increase the proportion of cells with aberrant chromosomes. The test item was thus not clastogenic in this in vitro test system under the conditions described.

Executive summary:

Study Design

The test material was investigated in three 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 endoreduplications 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:4
    others: 2
No. of metaphases per slide: 100 (structural abberations)
No. of metaphases per slide: 1000 (polyploidy)
Preparation times:
    -S9 mix: 25, 31 hours
    +S9 mix: 25 hours
Exposure times:
    -S9 mix: 5, 25, 31 hours
    +S9 mix: 5 hours
Solvent: acetone
Concentrations evaluated:
2.81, 8.89, and 28.1 µg/mL
Positive controls: EMS and Griseofulvin (-S9) and Cyclophosphamide (+S9)

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 473.

Results

The positive control 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 of polyploid cells.

The test item precipitated in the culture medium and induced cytotoxic effects, i.e. reduction in mitotic index or cell viability (MTT-test); both at concentrations of ≥15.8 µg/mL.
Thus, the limiting factors for the highest test concentration were cytotoxicity and solubility of the test material.
The mean values of aberrant metaphases (gaps excluded) in the negative controls of both experiments (- and + S9 mix) ranged from 0.5 % to 1.3 % and thus met the historical control range. The test item did not show any relevant increase in the number of  aberrant metaphases. Furthermore, no treatment-related increase in  endoreduplications or polyploid cells was observed. I.e. neither structural nor numerical aberrations were detected.

Conclusion

The treatment of V79 cell cultures with the test material did not increase the proportion of cells with aberrant chromosomes. The test item was thus not c1astogenic 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

Based on the provided information there is no need to classified according to the EU Regulation (EC) No 1 272/2008 on Classification,Labelling and Packaging of Substances and Mixtures.

Justification for classification or non-classification

Based on the provided information there is no need to classify according to the EU Regulatio n (EC) No 1272/2008 on Classification,Labelling and Packaging of Substances and Mixtures.