Benzyltoluene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2012-06-29 - 2012-12-19

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The GLP study was conducted according to an internationally accepted guideline. All study parameters are given in detail. Nevertheless, according to the ECHA's practical guide 6: "How to report read-across and categories" the maximum for read-cross is 2.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Phenyl-tolyl-ethane

IUPAC Name:

Phenyl-tolyl-ethane

Constituent 2

Reference substance name:

40766-30-1

Cas Number:

40766-30-1

IUPAC Name:

40766-30-1

Test material form:

other: liquid

Details on test material:

Name PTE
Batch no. Trailer
Appearance Clear Liquid
Composition 87.5% phenyl-tolyl-ethane (PTE)
10.8% 1,1-diphenylethane (DPE)
CAS No. 40766-30-1
EINECS-No. not stated
Molecular formula C15H16
Molecular weight 196 g/mol
Purity 87.5% PTE (GC)
Homogeneity homogeneous
Vapour pressure 0.0025 hPa at 20°C
Stability unknown
Solubility H2O: < 0.1g/L; acetone; CH3CN: > 1 g/L;
EtOH, DMSO: unknown
Storage Room Temperature: (20 ± 5°C)

Method

Target gene:

Autosomal thymidine kinase (TK) locus of heterozygous L5178Y/TK+/- cells

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

other: deficient in thymidine kinase

Metabolic activation:

with and without

Metabolic activation system:

liver enzyme S9 fraction

Test concentrations with justification for top dose:

In the first experiment, the following concentrations (real) of the test item were used:
963 µg/mL, 481.5 µg/mL, 240.8 µg/mL, 120.4 µg/mL, 60.2 µg/mL, 30.1 µg/mL, 15.0 µg/mL and 7.5 µg/mL.
In the second experiment, the following concentrations (real) of the test item were used:
488.8 µg/mL, 244.4 µg/mL, 122.2 µg/mL, 61.1 µg/mL, 30.5 µg/mL, 15.3 µg/mL, 7.6 µg/mL and 3.8 µg/mL.

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Details on test system and experimental conditions:

For mycoplasma contamination screened stocks of cells are stored in liquid nitrogen in the cell bank to allow a continuous stock of cells, which guarantees similar parameters of the experiment and reproducible characteristics of the cells.
The L5178Y cells are thawed 120 hours prior to treatment and cultivated in RPMI 1640 complete culture medium (with 5% horse serum) in cell culture flasks (75 cm2)and incu-bated at 37.0 ± 1.5 °C in a humidified atmosphere with 5% CO2.

Evaluation criteria:

The test item is considered to have mutagenic effects if:
 the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 106 cells above the corresponding solvent control.
 the relative increase of the mutation frequency shows a dose relationship.
A mutagenic response is considered to be reproducible if it occurs in both parallel cul-tures.
Results of test groups are generally rejected if the relative total growth is less than 10% of the solvent control.
The biological relevance of the results is always considered first. Appropriate statistical methods are used as an aid in evaluating the test results. However, the results of statisti-cal testing were assessed with respect to dose-response relationship. Reproducibility and historical data was also taken into consideration.
Statistical significance was confirmed by means of the non-parametric χ2 test. However, both biological and statistical significance were considered together.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Treatment	Conc. (µg/ml)	Relative Total Growth Culture A	Mutants per 10^6cells Culture A	Relative Total Growth Culture B	Mutants per 10^6cells Culture B	Relative Total Growth Mean	Mutants per 10^6cells Mean
Exp. I with metabolic activation (S9), exposition time 4 hours
Negative control	--	--	173	--	103	--	138
Solvent control DMSO	5 µL/mL	--	124.5	--	140	--	132.25
Solvent control 0.9% NaCl	5 µL/mL	--	140	--	142.5	--	141.3
Positive control CPA	4.5	27.6%	551	61.3%	570.5	44.5%	560.75
Test Item	963	0.1%	--	1.0%	360	0.5%	180
Test Item	482	0.0%	--	0.0%	--	0.0%	--
Test Item	241	5.0%	255	6.0%	230	5.5%	242.5
Test Item	120	51.6%	140	68.9%	102.5	60.2%	121.25
Test Item	60	70.0%	147.5	74.9%	156	72.5%	151.75
Test Item	30	90.4%	125.5	86.1%	168	88.2%	146.75
Test Item	15	78.7%	161.5	90.8%	160	84.8%	160.75
Test Item	7.5	122.9%	109.5	107.4%	158.5	115.1%	134
Exp. I without metabolic activation, exposition time 4 hours
Negative control	--	--	249.5	--	236	--	242.75
Solvent control DMSO	5 µL/mL	--	229.5	--	160.5	--	195
Positive control MMS	19.5	40.0%	581	54.3%	592.5	47.1%	586.75
Test Item	963	0.0%	--	0.1%	--	0.0%	--
Test Item	482	0.0%	--	0.0%	--	0.0%	--
Test Item	241	1.1%	581	1.4%	894	1.2%	737.5
Test Item	120	44.5%	232	64.5%	270	54.5%	251
Test Item	60	109.0%	141	97.6%	244.5	103.3%	192.75
Test Item	30	86.1%	172	107.9%	264.5	97.0%	218.25
Test Item	15	107.3%	214	94.0%	315.5	100.6%	264.75
Test Item	7.5	134.4%	129.5	119.6%	212	127.0%	170.75

Values above threshold (see below) are given in bold characters.

Threshold for mutagenic effects: number of mutant colonies per 10⁶cells of the respective solvent control plus 126.

Threshold negative contr. (medium) with metabolic activation:       264 mutants/10⁶cells

Threshold negative contr. (medium) without metabolic activation: 368.75 mutants/10⁶cells

Threshold solvent control NaCl 0.9% with metabolic activation:     267.3 mutants/10⁶cells

Threshold solvent control DMSO with metabolic activation:256.3 mutants/10⁶cells

Threshold solvent control DMSO without metabolic activation:      321 mutants/10⁶cells

Treatment	Conc. (µg/ml)	Relative Total Growth Culture A	Mutants per 10^6cells Culture A	Relative Total Growth Culture B	Mutants per 10^6cells Culture B	Relative Total Growth Mean	Mutants per 10^6cells Mean
Exp. II with metabolic activation (S9), exposition time 4 hours
Negative control	--	--	173.5	--	139.5	--	156.5
Solvent control DMSO	5 µL/mL	--	138	--	101	--	119.5
Solvent control 0.9% NaCl	5 µL/mL	--	156.5	--	135.5	--	146
Positive control CPA	4.5	62.3%	536.5	45.1%	659	53.7%	597.75
Test Item	488	0.0%	--	0.0%	--	0.0%	--
Test Item	244	8.9%	188.5	8.9%	269	8.9%	228.75
Test Item	122	66.3%	201.5	71.0%	193.5	68.7%	197.5
Test Item	61	73.9%	210.5	62.4%	269.5	68.2%	240
Test Item	31	73.6%	176.5	86.4%	201	80.0%	188.75
Test Item	15	85.2%	187.5	106.7%	239.5	96.0%	213.5
Test Item	7.6	69.7%	235	89.0%	245	79.3%	240
Test Item	3.8	75.5%	231	98.6%	233	87.1%	232
Exp. II without metabolic activation, exposition time 24 hours
Negative control	--	--	128.5	--	139	--	133.75
Solvent control DMSO	5 µL/mL	--	152	--	125.5	--	138.75
Positive control MMS	19.5	14.7%	1341.5	12.1%	1363	13.4%	1352.25
Test Item	488	0.0%	--	0.0%	--	0.0%	--
Test Item	244	0.7%	239.5	0.4%	520.5	0.5%	380
Test Item	122	73.9%	231	59.3%	282.5	66.6%	256.75
Test Item	61	113.6%	195	86.6%	245.5	100.1%	220.5
Test Item	31	94.2%	226	97.4%	231	95.8%	228.5
Test Item	15	96.3%	208.5	91.0%	202	93.7%	205.25
Test Item	7.6	91.5%	165.5	79.8%	189	85.6%	177.25
Test Item	3.8	92.0%	216.5	109.7%	243	100.8%	229.75

Values above threshold (see below) are given in bold characters.

Threshold for mutagenic effects: number of mutant colonies per 10⁶cells of the respective solvent control plus 126.

Threshold negative contr. (medium) with metabolic activation:       382.5 mutants/10⁶cells

Threshold negative contr. (medium) without metabolic activation: 259.75 mutants/10⁶cells

Threshold solvent control NaCl 0.9% with metabolic activation:     272 mutants/10⁶cells

Threshold solvent control DMSO with metabolic activation:245.5 mutants/10⁶cells

Threshold solvent control DMSO without metabolic activation:      264.75 mutants/10⁶cells

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Therefore, PTE is considered to be non-mutagenic under the conditions of the mouse lymphoma assay.

Executive summary:

This study was performed to investigate the potential of PTEto induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The assay was performed in two independent experiments, using two parallel cultures each (replicates). The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed using a 4 h treatment period with metabolic activation and a 24 h treatment period without metabolic activation.

MMS (19.5 µg/mL) and CPA (4.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies and an increase of the relative quantity of small versus large induced colonies.In the first experiment, eight concentrations (ranging from 963 to 7.5 µg/mL) of the test item soluble in DMSO were used and tested with and without metabolic activation (4 hours incubation).Precipitation of the test item was not observed. In non-toxic concentrations, no increase in mutation frequency could be detected.

In the second experiment, eight concentrations (ranging from 488.8 to 3.8 µg/mL) of the test item soluble in DMSO were used and tested with (4 hours incubation) and without metabolic (22 hours incubation) activation.Precipitation of the test item was not observed. In non-toxic concentrations, no increase in mutation frequency could be detected in the second experiment, either.

In the first and in the second experiment, in the treatment without metabolic activation, a minor dose-response relationship was found. But as all mutant frequencies in non-toxic concentrations lay below the threshold (solvent control plus 126 colonies/10⁶cells), this effect was considered as not relevant for the classification of the test item.

An increase in mutant colony numbers was observed in the both experiments in the treatment without metabolic activation: the concentration 245 µg/mL (nominal) showed an increase of the mutant frequency. This concentration showed a clear cytotoxic effect towards the cells, though (relative total growth was 1.2% resp. 0.5 %); therefore, the increase of the mututant colonies was considered as artefact.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and the presence of metabolic activation.