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

Endpoint summary

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) according to OECD guideline 471, 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. In a further reverse mutation assay according to OECD guideline 471, 1,1'-(1,1,2,2-tetramethylethylene)dibenzene was not mutagenic in E. coli strain WP2uvrA.

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 according to OECD guideline 476.

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 an In vitro mammalian chromosome aberration test in Chinese Hamster V79 cells according to OECD guideline 473.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2021-10-04 to 2022-01-26

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Only E. coli strain WP2uvrA was tested

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008-05-31

Deviations:

yes

Remarks:

Only E. coli strain WP2uvrA was tested as a suitable Bacterial Reverse Mutation Assay with Salmonella typhimurium strains TA 1535, 1537, 98 and 100 has already been conducted (see IUCLID section 7.6.1).

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

2020-06-26

Deviations:

yes

Remarks:

Only E. coli strain WP2uvrA was tested as a suitable Bacterial Reverse Mutation Assay with Salmonella typhimurium strains TA 1535, 1537, 98 and 100 has already been conducted (see IUCLID section 7.6.1).

Principles of method if other than guideline:

Only E. coli strain WP2uvrA was tested as a suitable Bacterial Reverse Mutation Assay with Salmonella typhimurium strains TA 1535, 1537, 98 and 100 has already been conducted (see IUCLID section 7.6.1).

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

The Escherichia coli WP2uvrA strain detects base-pair substitutions (tryptophan- to tryptophan+ reversions).

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: Rat liver S9 prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
- Method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 mL or 5.0 mL Milli-Q water (first or second experiment respectively); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution; 1 mL 0.33 M KCl solution. The above solution was filter (0.22 μm)-sterilized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 9.0 mL of S9-mix components 1.0 mL S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment.
- Concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL S9 mix (containing 5% S9 in experiment I and 10% S9 in experiment II) were contained in 3.7 mL of final culture medium.
- Quality controls of S9: Each S9 batch is characterized with the mutagens benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively.

Test concentrations with justification for top dose:

Experiment I (with and without S9): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
Experiment II (with and without S9): 86, 154, 275, 492, 878 and 1586 μg/plate.

The highest concentration of the test item used in experiment II was the level at which the test item exhibited limited solubility.

Vehicle / solvent:

- Vehicles used: THF (test item), DMSO (4-NQO, 2AA)

- Justification for choice of solvent/vehicle: A solubility test was performed based on visual assessment. The test item proved insoluble in Milli-Q Water, DMSO, ethanol, acetone and hexane, but formed a clear colourless solution in THF.

- Justification for percentage of solvent in the final culture medium: The percentage of solvent in the final culture medium was 1.35%. It is known from the historical control database of the laboratory that this concentration is non-toxic to bacteria.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Tetrahydrofuran (test item), DMSO (4-NQO, 2AA)

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

other: 2-aminoanthracene

Remarks:

With S9
2AA: 15 µg/plate

Without S9
4-NQO: 10 µg/plate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 10^9 cells/mL
- Incubation method: Plate incorporation method (experiment I and II)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 ± 4 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Reduction of the bacterial background lawn, increase in the size of the microcolonies and the reduction of the revertant colonies

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Number of revertants

Rationale for test conditions:

In the first experiment the test item precipitated on the plates at dose levels of 521 μg/plate and upwards. Since the test item precipitated heavily on the plates at the highest concentration in the presence of S9-mix, the number of revertants of this dose level could not be determined. In the second experiment the test item precipitated on the plates at dose level of 275 μg/plate and upwards in the absence of S9-mix and at dose level 492 and upwards in de presence of S9-mix. No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed at the tested concentrations.

Thus, concentration specified under "Test concentrations with justification for top dose" were evaluated.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain WP2uvrA is not greater than two times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain WP2uvrA is greater than two times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed, the positive response should be reproducible in at least one follow up experiment.

Statistics:

No required according to OECD guideline 471.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

other: Not tested. This strain was tested in another Bacterial Reverse Mutation Assay (see IUCLID section 7.6.1)

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

not examined

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

other: Not tested. This strain was tested in another Bacterial Reverse Mutation Assay (see IUCLID section 7.6.1)

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

not examined

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

other: Not tested. This strain was tested in another Bacterial Reverse Mutation Assay (see IUCLID section 7.6.1)

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

not examined

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

other: Not tested. This strain was tested in another Bacterial Reverse Mutation Assay (see IUCLID section 7.6.1)

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

not examined

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item proved insoluble in Milli-Q Water, DMSO, ethanol, acetone and hexane, but formed a clear colourless solution in THF.
- Precipitation and time of the determination: The test item precipitated on the plates at dose
levels of 521 μg/plate and upwards in experiment I and at dose level of 275 μg/plate and upwards in the absence of S9-mix and at dose level 492 and upwards in de presence of S9-mix in experiment II.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See "Attached background material"

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible: No concentration-response relationship was observed

Ames test:
- Signs of toxicity: No signs of toxicity were observed
- Individual plate counts: See "Attached background material"
- Mean number of revertant colonies per plate and standard deviation: See "Attached background material"

HISTORICAL CONTROL DATA
- Positive historical control data: See "Attached background material"
- Negative (solvent/vehicle) historical control data: See "Attached background material"

Conclusions:

In a Bacterial Reverse Mutation Assay according to OECd guideline 471, it is concluded that 1,1’-(1,1,2,2- tetramethylethylene)dibenzene is not mutagenic in Escherichia coli (E. coli) strain WP2uvrA.

Executive summary:

The objective of this study was to determine the potential of 1,1’-(1,1,2,2-tetramethylethylene)dibenzene and/or its metabolites to induce reverse mutations at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9). The study procedures described in this report were based on the most recent OECD 471 and EC B.13/14 guidelines. The vehicle of the test item was tertrahydrofuran. In the first experiment, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix. The test item precipitated on the plates at dose levels of 521 μg/plate and upwards. Since the test item precipitated heavily on the plates at the highest concentration in the presence of S9-mix, the number of revertants of this dose level could not be determined. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. In the second mutation experiment, the test item was tested at a concentration range of 86 to 1568 μg/plate in the absence and presence of 10% (v/v) S9-mix. The test item precipitated on the plates at dose level of 275 μg/plate and upwards in the absence of S9-mix and at dose level 492 and upwards in de presence of S9-mix. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. The test item did not induce a dose-related increase in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. In conclusion, based on the results of this study it is concluded that 1,1’-(1,1,2,2- tetramethylethylene)dibenzene is not mutagenic in the Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

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

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted May 26, 1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

84/449/EEC

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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.

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

other: Not tested. This strain was tested in another Bacterial Reverse Mutation Assay (see IUCLID section 7.6.1)

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

not examined

Key result

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

Key result

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

Key result

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

Key result

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.

Reference 3

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

Guideline:

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

Version / remarks:

July 21st 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 30th 2008

Deviations:

no

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

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.

Reference 4

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

Guideline:

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

Version / remarks:

31 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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.

Reference 5

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

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

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.

Reference 6

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

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

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:

- WoE study 1

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.

 

- WoE study 2

The objective of this study was to determine the potential of 1,1’-(1,1,2,2-tetramethylethylene)dibenzene and/or its metabolites to induce reverse mutations at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9). The study procedures described in this report were based on the most recent OECD 471 and EC B.13/14 guidelines. The vehicle of the test item was tertrahydrofuran. In the first experiment, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix. The test item precipitated on the plates at dose levels of 521 μg/plate and upwards. Since the test item precipitated heavily on the plates at the highest concentration in the presence of S9-mix, the number of revertants of this dose level could not be determined. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. In the second mutation experiment, the test item was tested at a concentration range of 86 to 1568 μg/plate in the absence and presence of 10% (v/v) S9-mix. The test item precipitated on the plates at dose level of 275 μg/plate and upwards in the absence of S9-mix and at dose level 492 and upwards in de presence of S9-mix. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. The test item did not induce a dose-related increase in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. In conclusion, based on the results of this study it is concluded that 1,1’-(1,1,2,2- tetramethylethylene)dibenzene is not mutagenic in the Escherichia coli 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 eighteenth time in Regulation (EU) 2022/692.