4-Ethoxy-4'-(trans-4-ethylcyclohexyl)-2,3-difluor-1,1'-biphenyl

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb 03, 2006 - Jun 22, 2006

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 guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997-07-21

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

4-ethoxy-2,3-difluoro-4'-[(1s,4r)-4-ethylcyclohexyl]-1,1'-biphenyl

EC Number:

608-727-0

Cas Number:

323178-01-4

Molecular formula:

C₂₂H₂₆F₂O

IUPAC Name:

4-ethoxy-2,3-difluoro-4'-[(1s,4r)-4-ethylcyclohexyl]-1,1'-biphenyl

Test material form:

solid

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: originally received from Hoffmann-La Roche, Pharma, Basel, on May 27, 1997
- Cell cycle length, doubling time or proliferation index: 16 to 17.5 hours
V79 cells have been successfully used in mutagenicity testing for many years. This cell line has a high proliferation rate and cloning efficiency. The cells have a relatively stable karyotype with a modal chromosome number of 22 ± 1, and an aberration rate of about 0-5 % of the metaphases. Since the cell line is not able to metabolize indirect mutagens to reactive forms, the test is performed both in the presence and absence of an external metabolizing system (liver S9 mix of rats pre-treated with Aroclor 1254).

Metabolic activation:

with and without

Metabolic activation system:

S9-mix with S9 from Aroclor 1254 induced rats

Test concentrations with justification for top dose:

In the first experimental series, test material concentrations ranging from 0.50 to 500 µg/mL were tested. The test material precipitated in the culture medium at concentrations ≥158 µg/mL in the absence and presence of S9 mix. No cytotoxic effects were seen. Quality control of the cell preparations did not show a relevant influence of the test material on the structure or spreading of the chromosomes in the concentration range investigated. A change in the pH or the osmotic value of the cell culture medium did not occur in the dose range tested.
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:
1st series: 15.8, 50.0, and 158 µg/mL
2nd series: 50.0, 88.9, 158, 281 and 500 µg/mL

Vehicle / solvent:

Acetone, 0.1 %

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

other: Griseofulvin

Details on test system and experimental conditions:

No. of slides per concentration:
Solvent control: 4
Others: 2
No. of metaphases evaluated per slide: 100 (for structural aberrations); 1000 (for polyploidy)

Preparation times: 25 and 35 hours

Exposure times:
- S9 mix: 5, 25 and 35 hours
+ S9 mix: 5 hours

Solvent for the test material: Acetone

Concentrations evaluated:
Test item first series: 15.8, 50.0 and 158 µg/mL
second series: 50.0, 88.9, 158, 281 and 500 µg/mL

Positive controls:
- S9 mix: 250 and 500 µg Ethylmethansulfonat (EMS)/mL
31.6 and 88.9 µg Griseofulvin (GRIS)/mL
+S9 mix: 2.00 µg Cyclophosphamide (CPA)/mL

Rationale for test conditions:

Guideline settings are applied

Evaluation criteria:

Step 1: Evaluation of Slide Quality
Step 2: Evaluation of Chomosomal Aberrations

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

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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 applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

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.

Executive summary:

The test item was investigated in two experimental series for induction of chromosomal aberrations in V79 Chinese hamster cells in vitro according to OECD 473. This also included examinations on whether or not the test material may have the potential to induce numerical aberrations, i.e. an increase in number of polyploid cells.

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); 1000 (for polyploidy)

Preparation times: 25 and 35hours

Exposure times: - S9 mix: 5, 25 and 35 hours; + S9 mix: 5 hours

Solvent for the test material: Acetone

Concentrations evaluated: Test item first series: 15.8, 50.0 and 158 µg/mL and second series: 50.0, 88.9, 158, 281 and 500 µg/mL

Positive controls:- S9 mix: 250 and 500 µg Ethylmethansulfonat (EMS)/mL, 31.6 and 88.9 µg Griseofulvin (GRIS)/mL and +S9 mix: 2.00 µg Cyclophosphamide (CPA)/mL

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.

The test item precipitated in the culture medium at concentrations of 158 µg/mL in the absence and presence of S9-mix. No cytotoxic effects were observed.

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 0.75 to 2.25 %. Treatment with the test item did not result in any biologically or statistically relevant increase in the number of aberrant metaphases in two independent experimental series at concentrations ranging from 15.8 to 500 µg/mL, except a slight but statistically significant increase at 281 µg/mL in the second experiment in the presence of S9 after 5+20 hour treatment. However, this effect did not occur at 500 µg/mL and is thus not concentration dependent. Furthermore, the mean number of aberrant metaphases was well within the range of historical control values of this laboratory. Therefore, this effect is considered incidental and of no biological relevance.

In the first experimental series an increase in the number of polypoid cells was observed in cultures treated with the test item in the presence of S9 at 158 µg/mL (5+20 hour treatment). However, in the second experiment there was no increase in the number of polypoid cells at test item concentrations ranging from 50.0 to 500 µg/mL after 5+20 hour treatment or 5+30 hour treatment. Thus, this effect was only observed in 1 of 3 experiments. Moreover, the detection of numerical aberrations is not the primary endpoint of this assay, therefore these observations are of questionable biological relevance.

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.