Benzyltoluene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative in all tests conducted (for details, see below).

Link to relevant study records

Reference

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.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

Target gene:

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

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

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.

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'.

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Available information:

Mutagenicity in vitro in bacteria:

1,1 -DPE:

A distillate containing 75 % diphenyl ethane, was tested for mutagenicity in the Ames test according to OECD guideline 471 using 5 strains of Salmonella typhimurium (TA 1535, TA 1537, TA 1538, TA 98 and TA 100).

The test item produced no significant increase in revertants in any strain and was considered non-mutagenic in this test.

SAS-40:

SAS-40 was tested in the Salmonella typhimurium reverse mutation assay according to OECD/EC guidelines with five histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA98 and TA100) with and without metabolic activation (S9 -mix). Based on the results of this study it can be concluded that SAS-40 is not mutagenic in the Salmonella typhimurium reverse mutation assay.

PTE:

Phenyl-tolyl-ethane was tested in the Salmonella typhimurium reverse mutation assay according to OECD/EC guidelines with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in tester strain WP2uvrA with and without metabolic activation (S9 -mix). Phenyl-tolyl-ethane did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation.

In vitro chromosome aberration:

1,1 -DPE:

XZ 95510, a distillate containing 75% diphenylethane (DPE), was tested for clastogenic potential using Chinese hamster ovary (CHO) cells -in vitro according to OECD guideline 473 (In vitro Mammalian Chromosome Aberration Test). Cell cultures were exposed to XZ 95510 at concentrations of 5, 10, 20 and 40 ug/ml , both in the presence and absence of a metabolic activation system (Aroclor- 1254 induced rat liver S9-mix) . There was no reproducible evidence that XZ 95510 induced chromosome aberrations and there was no indication of chromosomal ploidy changes in either the presence or absence of the metabolic activation system.

PTE:

A study according to OECD guideline 473 was performed to assess the effect of Phenyl-tolyl-ethane on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). Phenyl-tolyl-ethane was not clastogenic in human lymphocytes under the experimental conditions described.

Mouse Lymphoma Assay:

PTE:

This study according to OECD guideline 476 was performed to investigate the potential of PTE to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

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.

DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro

A sample of 99% diphenyl ethane was tested for genotoxicity by measuring unscheduled DNA synthesis (U.D. S.) in primary rat hepatocytes in -vitro according to OECD guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro). None of the concentrations of 99% diphenyl ethane showed any evidence of increase DNA labelling. It was concluded that 99% diphenyl ethane showed no evidence of genotoxicity in this U. D. S. assay.

Genetic toxicity in vivo:

Micronucleus Assay:

SAS-40:

In a mouse micronucleus test following OECD-guideline 474 under GLP conditions, SAS-40 was administered intraperitoneally at the maximun tolerated dose (400 mg/kg bw).

SAS-40 did not significantly increase the number of micronucleated polychromatic erythrocytes or significantly decrease the ratio of polychromatic to normochromatic erythrocytes at any of the time points. It can be concluded that SAS-40 does not induce micronuclei.

Conclusion:

All in vitro and in vivo tests in genetic toxicity showed negative results. There is no reason to believe that the negative results would not be relevant to humans. No further testing is required.

Justification for selection of genetic toxicity endpoint
Conclusion based on the following assays: Bacterial reverse mutation assay (Ames test); Mammalian cell gene mutation assay; In vitro mammalian chromosome aberration test;DNA Damage and Repair (Unscheduled DNA Synthesis in Mammalian Cells In Vitro); in vivo micronucleus assay

Justification for classification or non-classification

Based on the available data, no classification is needed.