1,1'-(1,1,2,2-tetramethylethylene)dibenzene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2011-11-30 to 2012-03-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

July 21st 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

May 30th 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

August 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

1,1'-(1,1,2,2-tetramethylethylene)dibenzene

EC Number:

217-568-2

EC Name:

1,1'-(1,1,2,2-tetramethylethylene)dibenzene

Cas Number:

1889-67-4

Molecular formula:

C18H22

IUPAC Name:

(2,3-dimethyl-3-phenylbutan-2-yl)benzene

Method

Target gene:

hypoxanthine-guanine phosphoribosyl transferase enzyme (hprt) locus located on the X chromosome

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham´s F12 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix (phenobarbital and ß-naphthoflavone induced rat liver)

Test concentrations with justification for top dose:

Experiment 1, 5-hour treatment period without S9 mix:
2.5, 5, 10, 15, 20, 25, 30, 35* and 40* μg/mL
Experiment 1, 5-hour treatment period with S9 mix:
5, 10, 15, 20, 25, 30, 35, 40 and 45 μg/mL
Experiment 2, 20-hour treatment period without S9 mix:
2.5, 5, 10, 15, 20, 22.5, 25, 27.5* and 30* μg/mL
Experiment 2, 5-hour treatment period with S9 mix:
2.5, 5, 10, 15, 20, 25, 30, 35, 40 and 45 μg/mL
Confirmatory Experiment, 5-hour treatment period without S9 mix:
2.5, 5, 10, 15, 20, 25, 30, 35* and 40* μg/mL

*These concentrations were very toxic and there were not enough cells to start the phenotypic expression period after the treatment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: N,N-dimethylformamide (DMF)

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Pre-incubation period: 24 hours
- Exposure duration: Experiment 1: 5 hours with and without S9 mix; Experiment 2: 20 h without S9 mix, 5 h with S9 mix; Confirmatory Experiment: 5 h without S9 mix
- Expression time (cells in growth medium): 19 hours in fresh F12 medium
- Selection time (if incubation with a selection agent): At the end of the expression period the cultures from each of the dose levels were aliquoted in 2x105 cells per 100-mm dish (five dishes) in selection medium (EX-CELL® CD CHO Serum-Free Medium for CHO Cells-SEL) containing 10 μM/mL of 6-thioguanine (6-TG).
- Fixation time (start of exposure up to fixation or harvest of cells): After the selection period, the colonies were fixed, stained with Giemsa and counted for mutant selection and cloning efficiency determination.

SELECTION AGENT: 6-thioguanine

NUMBER OF REPLICATIONS: Duplicate cultures were used at each concentration.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency: Toxicity was determined by comparing the colony forming ability of the treated groups to the negative (solvent) control and results were noted as percentage of cells in relation to the negative control.

Evaluation criteria:

The test item would have been considered to be mutagenic in this assay if all the following criteria were met:
• The assay is valid.
• The mutant frequency at one or more doses is significantly greater than that of the relevant control.
• Increase of the mutant frequency is reproducible.
• There is a clear dose-response relationship.
The test item would have been considered to have shown no mutagenic activity if no increases were observed which met the criteria listed above.

Statistics:

Statistical analysis was done with SPSS PC+ software for the following data:
• mutant frequency between the negative (solvent) and the test item or positive control item treated groups.
The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity is detected, a one-way analysis of variance was carried out. If the obtained result is positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity is found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there is a positive result, the inter-group comparisons are performed using the Mann-Whitney U-test.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Precipitation: no

RANGE-FINDING/SCREENING STUDIES:
Treatment concentrations for the mutation assay were selected on the basis of the result of a Pre-test on cell toxicity. A dose selection (cytotoxicity assay) was performed. During the cytotoxicity assay, 1-3 day old cultures (more than 50 % confluent) were trypsinised and cell suspensions were prepared in Ham's F12 medium. Cells were seeded into 90 mm petri dishes (tissue culture quality: TC sterile) at 10 E6 cells each and incubated in culture medium. After 24 hours the cells were treated with the suitable concentrations of the test item in absence or in presence (10 and 10 concentrations) of S9 mix (50 μL/mL) and incubated at 37 °C for 5 hours. After the treatment the cells were washed and incubated in fresh Ham's F12 medium for 19 hours. Additional groups of cells were treated for 20 hours without metabolic activation (10 concentrations). 24 hours after the beginning of treatment, the cultures were washed with Ham's F12 medium covered with trypsin-EDTA solution and counted and the cell concentration was adjusted to 40 cells/mL with Ham's F12 medium. For each dose, 5 mL was plated in parallel into 3 sterile dishes (diameter is approx. 60 mm). The dishes were incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air for 5-7 days for colony growing. Colonies were then fixed with methanol, stained with Giemsa and the colonies were counted. Survivals were assessed by comparing the colony forming ability of the treated groups to the negative (solvent) control. Precipitation of the test item in the final culture medium was examined visually at the beginning and end of the treatments. In addition, pH and osmolality were considered for dose level selection. Results of the Pre-test on cell toxicity were used for dose selection of the test item used in the Main Mutation Assays. 9 to 10 concentrations were selected for the treatment in Experiment 1 and 2.

COMPARISON WITH HISTORICAL CONTROL DATA: The mutant frequency in the negative (solvent) control cultures is within the range (min-max) of historical laboratory control data.

Remarks on result:

other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:

The test item 1,1'-(1,1,2,2-tetramethylethylene)dibenzene was not mutagenic in this in vitro cell gene mutation test performed with CHO-K1 (Chinese hamster ovary) cells.

Executive summary:

1,1`-(1,1,2,2 -tetramethylethylene)dibenzene was tested in a Mammalian Gene Mutation Test in CHO-K1 cells according to OECD guideline 476 and EU method B.17. The test item was dissolved in N,N-dimethylformamide and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation using S9 mix). Two independent main experiments (both run in duplicate) were performed at the concentrations and treatment intervals given below:

Experiment 1, 5-hour treatment period without S9 mix:

2.5, 5, 10, 15, 20, 25, 30, 35* and 40* μg/mL

Experiment 1, 5-hour treatment period with S9 mix:

5, 10, 15, 20, 25, 30, 35, 40 and 45 μg/mL

Experiment 2, 20-hour treatment period without S9 mix:

2.5, 5, 10, 15, 20, 22.5, 25, 27.5* and 30* μg/mL

Experiment 2, 5-hour treatment period with S9 mix:

2.5, 5, 10, 15, 20, 25, 30, 35, 40 and 45 μg/mL

Confirmatory Experiment, 5-hour treatment period without S9 mix:

2.5, 5, 10, 15, 20, 25, 30, 35* and 40* μg/mL

*: These concentrations were very toxic and there were not enough cells to start the phenotypic expression period after the treatment.

In Experiment 1, there were statistically significant increases (p < 0.01) in mutation frequency at concentration of 30 μg/mL in the absence of metabolic activation. In this Experiment, there were slightly higher mutation frequencies at the examined concentrations of 25 and 30 μg/mL in the presence of metabolic activation compared to the concurrent control. These alterations were statistically significant (p < 0.05). In Experiment 1 dose-response relationships were not noted in the absence and in the presence of metabolic activation.

In Experiment 2, the mutant frequency of the cells did not show statistically significant alterations compared to the concurrent control, when the test item was tested without S9 mix over a prolonged treatment period (20 hours). Furthermore, a five-hour treatment in the presence of S9 mix did not cause statistically significant increases in mutant frequency at concentrations of 25 and 30 μg/mL compared to the concurrent control, indicating that the findings in Experiment 1 were within the normal biological variation. A Confirmatory Experiment was necessary to conduct because of the equivocal findings in Experiment 1 (5-hour treatment period without metabolic activation).

In the Confirmatory Experiment, the mutant frequency of the cells did not show significant alterations compared to the concurrent control, indicating that the findings in Experiment 1 were within the normal biological variation. The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large increases in mutation frequency in the positive control cultures.

1,1`-(1,1,2,2 -tetramethylethylene)dibenzene tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this test in Chinese hamster ovary cells. The test item was not mutagenic in this in vitro mammalian cell gene mutation test performed with CHO-K1 cells.