Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test:
Under the experimental conditions of a reverse mutation assay (Ames test), 1,1'-(1,1,2,2-tetramethylethylene)dibenzene did not induce point mutations by base pair changes or frameshifts in the genome of four Salmonella typhimurium strains.

HPRT Test:
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.

In vitro mammalian chromosome aberration test in Chinese Hamster V79 cells:
1,1'-(1,1,2,2-tetramethylethylene)dibenzene is considered as not clastogenic in this system

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 1993-03-03 to 1993-03-25
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
only 4 bacterial strains investigated
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted May 26, 1983
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
84/449/EEC
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
The Salmonella typhimurium histidine (his) reversion system measures his- to his+ reversions.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from livers of Aroclor 1254 induced male rats
Test concentrations with justification for top dose:
33.3; 100.0; 333.3; 1000.0; 2500.0 and 5000.0 µg/plate
Vehicle / solvent:
- Vehicle used: acetone
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity for the bacteria.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation (S9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
without metabolic activation (S9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation (S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: at least 48 h at 37 °C in the dark

NUMBER OF REPLICATIONS: 3

NUMBER OF INDEPENDENT EXPERIMENTS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth, background lawn
Evaluation criteria:
A test article is considered as positive if either a dose-related and reproducible increase in the number of revertants or significant and reproducible increase for at least one test concentration is induced.
A test article producing neither a dose-related and reproducible increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
A significant response is described as follows:
A test article is considered as mutagenic if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537 and TA 98 it is at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.
Statistics:
No appropriate statistical method was available.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
To evaluate the toxicity of the test article a pre-study was performed with strains TA 98 and TA 100. The plates with the test article showed normal background growth up to 5000.0 µg/plate in both strains.

COMPARISON WITH HISTORICAL CONTROL DATA:
Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced revertant colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxic effects occurred in the test groups with and without metabolic activation in both independent experiments in all strains used. The plates incubated with the test article showed normal background growth up to 5000.0 mg/plate with and without S9 mix in all strains used.

MUTAGENICITY DATA:
No substantial increases in revertant colony numbers of any of the four tester strains were observed following treatment with the test item at any dose level in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher rates with increasing concentrations in the range below the generally acknowledged border of significance.
In the strains TA 1535 and TA 1537 a seemingly dose-dependent increase in revertant colony numbers were observed in experiment I without metabolic activation. In both strains the factor of 3 which is recommended for a mutagenic effect in these two strains could not be reached. However, the second experiment was carried out as a plate incorporation assay to examine whether the effects in the two strains could be reproduced. The results of the independent second experiment showed absolutely no tendency of a mutagenic effect in the above mentioned strains in the absence of metabolic activation. Therefore, the effect obtained in experiment I is considered not to be biologically relevant.
Remarks on result:
other: all strains/cell types tested
Conclusions:
Under the experimental conditions of a reverse mutation assay (Ames test), 1,1'-(1,1,2,2-tetramethylethylene)dibenzene did not induce point mutations by base pair changes or frameshifts in the genome of four Salmonella typhimurium strains.
Executive summary:

In a bacterial reverse mutation assay (Ames test) the mutagenic potential of 1,1'-(1,1,2,2-tetramethylethylene)dibenzene was investigated in the Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA1537 with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at 33.3; 100.0; 333.3; 1000.0; 2500.0 and 5000.0 µg/plate. The test was performed according to OECD 471 and Regulation (EC) 84/449/EEC Method B.13/14.

No toxic effects occurred in the test article groups with and without metabolic activation in two independent experiments in all strains used. The plates incubated with the test article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all strains used.

No substantial increases in revertant colony numbers of any of the four tester strains were observed following treatment with the test item at any dose level in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher rates with increasing concentrations in the range below the generally acknowledged border of significance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced revertant colonies.

In conclusion, it can be stated that under the experimental conditions of a reverse mutation assay (Ames test), 1,1'-(1,1,2,2-tetramethylethylene)dibenzene did not induce point mutations by base pair changes or frameshifts in the genome of four Salmonella typhimurium strains. Therefore, 1,1'-(1,1,2,2-tetramethylethylene)dibenzene is considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.

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
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
July 21st 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
May 30th 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
August 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
hypoxanthine-guanine phosphoribosyl transferase enzyme (hprt) locus located on the X chromosome
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)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
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.
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
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.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2012-06-11 until 2012-06-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
31 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
no target gene
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
This in vitro test is a cytogenetic test, which detects structural chromosome aberrations in somatic and/or germ cells and plays an important role in the evaluation of genotoxicity of a given item or agent (Preston et al., 1981). Structural aberrations develop due to breaks in one or both DNA strands, resulting in chromosome fragments (breaks, deletions). Faulty reunion of chromosome fragments results in formation of exchanges. These aberrations can be detected and quantified by light microscope. Extensive chromosome breaks usually cause cell death; small changes (breaks, deletions, translocations, inversions etc.) are, however, not necessarily lethal and can be regarded as an indication of molecular events, which might lead to malignant transformation.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver S 9 mix
Test concentrations with justification for top dose:
Experiment A with 3/20 h treatment/sampling time
without S9 mix: 10, 12.5, 15, 17.5, and 20* μg/mL

Experiment A with 3/20 h treatment/sampling time
with S9 mix: 12.5, 15, 17.5, 20, 22.51 and 25* μg/mL

Experiment B with 20/20 h treatment/sampling time
without S9 mix: 10, 12.5, 15 and 17.5 μg/mL

Experiment B with 20/28 h treatment/sampling time
without S9 mix: 10, 12.5, 15 and 17.5 μg/mL

Experiment B with 3/28 h treatment/sampling time
with S9 mix: 15, 17.5, 20, 22.5 and 25* μg/mL

* This concentration was tested but not evaluated because the lower concentrations were evaluated.
Vehicle / solvent:
N,N-dimethylformamide (DMF)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S-9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S-9 mix
Details on test system and experimental conditions:
The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes them suitable for gene toxicity assays with low background aberrations. These cells were chosen because of their small number of chromosomes (diploid number, 2n = 22) and because of the high proliferation rate (doubling time 12 - 14 h). The V79 cell line was established after spontaneous transformation of cells isolated from the lung of a normal Chinese hamster (male). This cell line was purchased from ECACC. The cell stocks were kept in a freezer at -80 °C. Checking of mycoplasma infections was carried out before freezing. Trypsin-EDTA (0.25 % Trypsin, 1 mM EDTA x 4 Na) solution was used for cell detachment to subculture. The laboratory cultures were maintained in 75 cm³ tissue culture (TC) plastic flasks at 37 °C in a humidified atmosphere containing 5 % CO2. The V79 cells for this study were grown in DME (Dulbecco’s Modified Eagle’s) medium supplemented with L-glutamine (2 mM) and 1 % of Antibiotic-antimycotic solution (containing 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and heat-inactivated foetal bovine serum (final concentration 10 %). During the 3 and 20 hours treatments with test item, solvent (negative control) and positive controls, the serum content was reduced to 5 %.

Experiment A:
The test item was dissolved in N,N-dimethylformamide for the treatment (stock solution: 10 mg/mL). The appropriate amount of this stock solution was diluted with medium to obtain the examination concentrations. Duplicate cultures were used at each concentration and the solvent control cultures as well as the positive controls for treatment without and with S9 mix. 5E05 cells were set up at each group. The culture medium of exponentially growing cell cultures was replaced with medium containing the test item. The exposure period was 3 hours. The exposure period followed by washing the cells with DME medium and then growth medium was added. Sampling was made at 20 hours (approximately 1.5 normal cell cycles from the beginning of treatment). For concurrent measures of cytotoxicity for all treated and negative control cultures, 5E05 cells were set up.

Experiment B:
The test item was dissolved in N,N-dimethylformamide for the treatment (stock solution: 10 mg/mL). The appropriate amount of this stock solution was diluted with medium to obtain the examination concentrations. In the cytogenetic Experiment B the exposure period without metabolic activation was 20 hours. The exposure period with metabolic activation was 3 hours. Experiment B, as Experiment A, included concurrent non-activated and S9-activated positive and negative controls. For each group 5E05 cells/dish were seeded. Sampling was made at 1.5 cell cycles (20 hours, without S9 mix only) and at approximately 2 normal cell cycles (28 hours, without and with S9 mix) from the beginning of treatment to cover a potential mitotic delay.
Evaluation criteria:
The criteria for determining a positive result are:
– a concentration-related increase or a reproducible increase in the number of cells with aberrations.
– biological relevance of the results should be considered first, however, for the interpretation of the data both biological and statistical significance should be considered together.
– an increase in the number of polyploid cells may indicate that the test item has the potential to inhibit mitotic processes and to induce numerical chromosome aberrations.
– an increase in the number of cells with endoreduplicated chromosomes may indicate that the test item has the potential to inhibit cell cycle progression.

A test item for which the results do not meet the above criteria is considered as non-mutagenic in this system
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:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The cytotoxicity at the highest concentrations was adequate in the studies (Experiments A and B) as indicated by a reduction of % cell survival to about 50 % or less.
In Experiment A, 1,1-(1,1,2,2 -tetramethylethylene)dibenzene did not induce an increase in the number of cells with aberrations at any examined concentration, either in the absence or in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistically significant differences between treatment and control groups and no dose-response relationship was noted.
In Experiment B, the test item was examined without S9 mix, over a long treatment period and the sampling was made at approximately 1.5 cell cycles (20 hours after treatment start). The cells with structural chromosome aberrations without gaps did not show significant alterations compared to the concurrent solvent controls. There was no increase in the number of cells with aberrations without S9 mix following exposure over a long treatment period of 20 hours and sampling at approximately 2 cell cycles (28 hours after treatment start).
A 3-hour treatment in the presence of S9 mix with 28-hour harvest from the beginning of treatment did not cause an increase in the number of cells with structural chromosome aberrations without gaps over concurrent solvent control.
As in Experiment A, in Experiment B no statistically significant differences between treatment and control groups and no dose-response relationships were noted. The observed chromosome aberration rates were within the ranges of historical control data.
No increase in the rate of polyploid and endoreduplicated metaphases was found after treatment with the different concentrations of the test item.
pH and osmolality values of control and treatment solutions were measured. In Experiments A and B no significant differences between treatment and control groups were observed.
In the concurrent solvent control group the percentage of cells with structural aberration(s) without gap was equal or less than 5 %, proving the suitability of the cell line used.
The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 μL/mL) and Cyclophosphamide (5 μg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations. Thus, the study is considered as valid.
Remarks on result:
other: all strains/cell types tested
Conclusions:
1,1`-(1,1,2,2 -tetramethylethylene)dibenzene is considered as not clastogenic in this system.
Executive summary:

1,1`-(1,1,2,2 -tetramethylethylene)dibenzene was tested in a Chromosome Aberration Assay in V79 cells according to OECD Guideline 473. The test item was dissolved in N,N-dimethylformamide (DMF) and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (with and without metabolic activation using S9 mix). In two independent experiments (both run in duplicate) at least 200 well-spread metaphase cells were analysed at concentrations and incubation/expression intervals given below, ranging from little to maximum (below 50 % survival) toxicity:

Experiment A with 3/20 h treatment/sampling time without S9 mix: 10, 12.5, 15, 17.5, and 20* μg/mL

Experiment A with 3/20 h treatment/sampling time with S9 mix: 12.5, 15, 17.5, 20, 22.5* and 25* μg/mL

Experiment B with 20/20 h treatment/sampling time without S9 mix: 10, 12.5, 15 and 17.5 μg/mL

Experiment B with 20/28 h treatment/sampling time without S9 mix: 10, 12.5, 15 and 17.5 μg/mL

Experiment B with 3/28 h treatment/sampling time with S9 mix: 15, 17.5, 20, 22.5 and 25* μg/mL

* This concentration was tested but not evaluated because the lower concentrations were evaluated.

In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, either in the absence or in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between treatment and concurrent solvent control groups and no dose-response relationships were noted. In Experiment B, the frequency of the cells with structural chromosome aberrations did not show significant alterations compared to concurrent controls, up to cytotoxic concentrations without S9 mix over a prolonged treatment period of 20 hours with harvest at 20 or 28 hours following treatment start. Further, a 3-hour treatment up to cytotoxic concentrations in the presence of S9 mix with 28-hour harvest from the beginning of treatment did not cause an increase in the number of cells with structural chromosome aberrations. In both experiments, no statistically significant differences between treatment and concurrent solvent control groups and no dose-response relationships were noted. The observed chromosome aberration rates were within the ranges of historical control data. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested. The validity of the test was shown using ethyl methanesulphonate (0.4 or 1.0 μL/mL) and Cyclophosphamide (5.0 μg/mL) as concurrent positive controls. Therefore, the test item is considered as not clastogenic in this system.

1,1'-(1,1,2,2-tetramethylethylene)dibenzene tested up to cytotoxic concentrations, both with and without mammalian metabolic activation system, did not induce structural chromosome aberrations in Chinese Hamster lung cells. Therefore, the test item is considered as not clastogenic in this system.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
from 1982-02-22 to 1982-02-26
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
not specified
Qualifier:
equivalent or similar to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Stock cultures of the six strains are stored in liquid nitrogen (-196 °C). Strains are regularly checked for their histidine requirement, sensitivity to UV radiation, crystal violet sensitivity, ampicillin resistance (TA97, TA98, and TA100) and the number of spontaneous revertants.
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
Stock cultures of the six strains are stored in liquid nitrogen (-196 °C). Strains are regularly checked for their histidine requirement, sensitivity to UV radiation, crystal violet sensitivity, ampicillin resistance (TA97, TA98, and TA100) and the number of spontaneous revertants.
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 97
Details on mammalian cell type (if applicable):
Stock cultures of the six strains are stored in liquid nitrogen (-196 °C). Strains are regularly checked for their histidine requirement, sensitivity to UV radiation, crystal violet sensitivity, ampicillin resistance (TA97, TA98, and TA100) and the number of spontaneous revertants.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from Aroclor 1254 induced rat livers
Test concentrations with justification for top dose:
20, 100, 500, 1000, 3000 µg/plate
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: insoluble in water, good solubility in DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation (S9 mix)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine
Remarks:
without metabolic activation (S9 mix)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation (S9 mix)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation (S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h at 37 °C
NUMBER OF REPLICATIONS: 3
NUMBER OF INDEPENDENT EXPERIMENTS: 1
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
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: yes at 500, 1000 and 3000 µg/plate

All bacterial strains gave negative responses over the whole dose range of the test substance. A clearing of the background lawn and a reduction in the number of spontaneous revertants occurred at a concentration of 3330 µg/plate, indicating that the test substance has been tested up to the lower limit of toxicity. Strain-specific positive control chemicals show that the test conditions have been optimal and the metabolic activation system (S9 mix) functioned properly. On the basis of the results, the test substance can be considered as non-mutagenic in the Ames Salmonella/microsome test.
Remarks on result:
other: all strains/cell types tested
Conclusions:
1,1'-(1,1,2,2-tetramethylethylene)dibenzene induced no statistically significant dose-related increase in the numbers of revertant (his+) colonies in each of the six tester strains. The test substance can, therefore, be considered as nonmutagenic in the Ames test.
Executive summary:

In a reverse gene mutation assay bacteria strains TA 1535, TA 1537, TA 1538, TA 97, TA 98 and TA 100 of S. typhimurium were exposed to 1,1'-(1,1,2,2-tetramethylethylene)dibenzene at concentrations of 20, 100, 500, 1000, 3000 µg/plate in the presence and absence of mammalian metabolic activation in a plate incorporation test.

All bacterial strains gave negative responses over the whole dose range of the test substance. A clearing of the background lawn and a reduction in the number of spontaneous revertants occurred at a concentration of 3330 µg/plate, indicating that the test substance has been tested up to the lower limit of toxicity. Strain-specific positive control chemicals show that the test conditions have been optimal and the metabolic activation system (S9 mix) functioned properly. On the basis of the results, the test substance can be considered as non-mutagenic in the Ames Salmonella/microsome test.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
from 1983-05-02 to 1983-05-06
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
not specified
Qualifier:
equivalent or similar to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Stock cultures of the six strains are stored in liquid nitrogen (-196 °C). Strains are regularly checked for their histidine requirement, sensitivity to UV radiation, crystal violet sensitivity, ampicillin resistance (TA97, TA98, and TA100) and the number of spontaneous revertants.
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 97
Details on mammalian cell type (if applicable):
Stock cultures of the six strains are stored in liquid nitrogen (-196 °C). Strains are regularly checked for their histidine requirement, sensitivity to UV radiation, crystal violet sensitivity, ampicillin resistance (TA97, TA98, and TA100) and the number of spontaneous revertants.
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
Stock cultures of the six strains are stored in liquid nitrogen (-196 °C). Strains are regularly checked for their histidine requirement, sensitivity to UV radiation, crystal violet sensitivity, ampicillin resistance (TA97, TA98, and TA100) and the number of spontaneous revertants.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from Aroclor 1254 induced rat livers
Test concentrations with justification for top dose:
10, 33, 100, 333, 1000 µg/plate
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: insoluble in water, good solubility in DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation (S9 mix)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine
Remarks:
without metabolic activation (S9 mix)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation (S9 mix)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation (S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h at 37 °C
NUMBER OF REPLICATIONS: 3
NUMBER OF INDEPENDENT EXPERIMENTS: 1
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
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: yes, at 333 and 1000 µg/plate

All bacterial strains showed negative responses over the whole dose range of the test substance. Strain-specific positive control chemicals showed that the test conditions were optimal and the metabolic activation system functioned properly. Based on the results, the test substance can be considered, as non-mutagenic in the Ames/microsome assay.
Remarks on result:
other: all strains/cell types tested
Conclusions:
1,1'-(1,1,2,2-tetramethylethylene)dibenzene induced no statistically significant dose-related increase in the numbers of revertant (his+) colonies in each of the five tester strains. The test substance can, therefore, be considered as non-mutagenic in the Ames test.
Executive summary:

In a reverse gene mutation assay bacteria strains TA 1535, TA 1537, TA 97, TA 98 and TA 100 of S. typhimurium were exposed to 1,1'-(1,1,2,2-tetramethylethylene)dibenzene at concentrations of 10, 33, 100, 333 and 1000 µg/plate in the presence and absence of mammalian metabolic activation in a plate incorporation test.

All bacterial strains showed negative responses over the whole dose range of the test substance. Strain-specific positive control chemicals showed that the test conditions were optimal and the metabolic activation system functioned properly. Based on the results, the test substance can be considered, as non-mutagenic in the Ames/microsome assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames test:

- Key study

In a bacterial reverse mutation assay (Ames test) the mutagenic potential of 1,1'-(1,1,2,2-tetramethylethylene)dibenzene was investigated in the Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA1537 with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at 33.3; 100.0; 333.3; 1000.0; 2500.0 and 5000.0 µg/plate. The test was performed according to OECD 471 and Regulation (EC) 84/449/EEC Method B.13/14.

No toxic effects occurred in the test article groups with and without metabolic activation in two independent experiments in all strains used. The plates incubated with the test article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all strains used.

No substantial increases in revertant colony numbers of any of the four tester strains were observed following treatment with the test item at any dose level in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher rates with increasing concentrations in the range below the generally acknowledged border of significance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced revertant colonies.

In conclusion, it can be stated that under the experimental conditions of a reverse mutation assay (Ames test), 1,1'-(1,1,2,2-tetramethylethylene)dibenzene did not induce point mutations by base pair changes or frameshifts in the genome of four Salmonella typhimurium strains. Therefore, 1,1'-(1,1,2,2-tetramethylethylene)dibenzene is considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.

- Supporting study 1

In a reverse gene mutation assay bacteria strains TA 1535, TA 1537, TA 1538, TA 97, TA 98 and TA 100 of S. typhimurium were exposed to 1,1'-(1,1,2,2-tetramethylethylene)dibenzene at concentrations of 20, 100, 500, 1000, 3000 µg/plate in the presence and absence of mammalian metabolic activation in a plate incorporation test.

All bacterial strains gave negative responses over the whole dose range of the test substance. A clearing of the background lawn and a reduction in the number of spontaneous revertants occurred at a concentration of 3330 µg/plate, indicating that the test substance has been tested up to the lower limit of toxicity. Strain-specific positive control chemicals show that the test conditions have been optimal and the metabolic activation system (S9 mix) functioned properly. On the basis of the results, the test substance can be considered as non-mutagenic in the Ames Salmonella/microsome test.

- Supporting study 2

In a reverse gene mutation assay bacteria strains TA 1535, TA 1537, TA 97, TA 98 and TA 100 of S. typhimurium were exposed to 1,1'-(1,1,2,2-tetramethylethylene)dibenzene at concentrations of 10, 33, 100, 333 and 1000 µg/plate in the presence and absence of mammalian metabolic activation in a plate incorporation test.

All bacterial strains showed negative responses over the whole dose range of the test substance. Strain-specific positive control chemicals showed that the test conditions were optimal and the metabolic activation system functioned properly. Based on the results, the test substance can be considered, as non-mutagenic in the Ames/microsome assay.

In vitro cell gene mutation (HPRT) test

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.

In vitro mammalian chromosome aberration test in Chinese Hamster V79 cells

1,1`-(1,1,2,2 -tetramethylethylene)dibenzene was tested in a Chromosome Aberration Assay in V79 cells according to OECD Guideline 473. The test item was dissolved in N,N-dimethylformamide (DMF) and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (with and without metabolic activation using S9 mix). In two independent experiments (both run in duplicate) at least 200 well-spread metaphase cells were analysed at concentrations and incubation/expression intervals given below, ranging from little to maximum (below 50 % survival) toxicity:

Experiment A with 3/20 h treatment/sampling time without S9 mix: 10, 12.5, 15, 17.5, and 20* μg/mL

Experiment A with 3/20 h treatment/sampling time with S9 mix: 12.5, 15, 17.5, 20, 22.5* and 25* μg/mL

Experiment B with 20/20 h treatment/sampling time without S9 mix: 10, 12.5, 15 and 17.5 μg/mL

Experiment B with 20/28 h treatment/sampling time without S9 mix: 10, 12.5, 15 and 17.5 μg/mL

Experiment B with 3/28 h treatment/sampling time with S9 mix: 15, 17.5, 20, 22.5 and 25* μg/mL

* This concentration was tested but not evaluated because the lower concentrations were evaluated.

In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, either in the absence or in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between treatment and concurrent solvent control groups and no dose-response relationships were noted. In Experiment B, the frequency of the cells with structural chromosome aberrations did not show significant alterations compared to concurrent controls, up to cytotoxic concentrations without S9 mix over a prolonged treatment period of 20 hours with harvest at 20 or 28 hours following treatment start. Further, a 3-hour treatment up to cytotoxic concentrations in the presence of S9 mix with 28-hour harvest from the beginning of treatment did not cause an increase in the number of cells with structural chromosome aberrations. In both experiments, no statistically significant differences between treatment and concurrent solvent control groups and no dose-response relationships were noted. The observed chromosome aberration rates were within the ranges of historical control data. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested. The validity of the test was shown using ethyl methanesulphonate (0.4 or 1.0 μL/mL) and Cyclophosphamide (5.0 μg/mL) as concurrent positive controls. Therefore, the test item is considered as not clastogenic in this system.

1,1'-(1,1,2,2-tetramethylethylene)dibenzene tested up to cytotoxic concentrations, both with and without mammalian metabolic activation system, did not induce structural chromosome aberrations in Chinese Hamster lung cells. Therefore, the test item is considered as not clastogenic in this system.

Justification for classification or non-classification

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

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