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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 10cells

Culture A

Relative Total Growth Culture B

Mutants per 10cells Culture B

Relative Total Growth Mean

Mutants per 10cells 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 10cells

Culture A

Relative Total Growth Culture B

Mutants per 10cells Culture B

Relative Total Growth Mean

Mutants per 10cells 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.