Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The target substance 1-Methyl-1-phenylethyl peroxyneodecanoate was tested in an in vitro genotoxicity testing battery as required by Annex VIII of the REACH regulation 1907/2006 (OECD 471, 473 and 476, GLP).

In a bacterial reverse gene mutation test conducted according to OECD 471, the target substance did not induce mutagenicity. In a mammalian cell HPRT mutation assay conducted according to OECD 476, the target substance 1-Methyl-1-phenylethyl peroxyneodecanoate was also tested negative for inducing mutagenic effects. Moreover, the target substance was tested negative in an in vitro cytogenicity study in human lymphocytes conducted according to OECD 473. Based on the lack of mutagenicity/cytogenicity in all in vitro assays, 1-Methyl-1-phenylethyl peroxyneodecanoate is considered to be non-genotoxic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2000-07-25 to 2000-12-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 July 1992
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
TEST MATERIAL
- Name: Cumyl peroxyneodecanoate
- Brand name: LUPEROX 188M70
- CAS-No.: 26748-47-0
- Purity: 70% (in isododecane)
- Appearance: brown liquid

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Source: ATOFINA. Cours Michelet, La Défense 10, 92091 Paris-la-Défense CEDEX, France; Batch no.: E-660 0005-001MXL
- Expiration date of the lot/batch: May 2001

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At -20 °C in the dark
- Solubility: 0.2 mg/L in water, miscible with organic solvents
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was dissolved in the vehicle at a concentration of 100 mg/mL (expressed as active material) for the preliminary toxicity test and both mutagenicity experiments. The preparations were made immediately before use.
Target gene:
histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: B.N. Ames' Laboratory (University of California, Berkeley, USA)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: stored in a cryoprotective medium (1 mL nutrient broth and 0.09 mL dimethyl sulfoxide) in a liquid nitrogen container.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Since the test substance was freely soluble and sometimes toxic, the highest dose-level was either 5000 µg/plate or based on the level of toxicity, according to the criteria specified in the international guidelines.

Experiments without S9 mix:
- 312.5, 625, 1250, 2500 and 5000 µg/plate, for TA1537, TA98, TA100 and TA102 (Experiment 1)
- 156.25, 312.5, 625, 1250 and 2500 µg/plate, for TA1535 (Experiment 1)
- 78.125, 156.25, 312.5, 625, 1250 and 2500 µg/plate, for all (Experiment 2)

Experiments with S9 mix:
- 312.5, 625, 1250, 2500 and 5000 µg/plate, for TA1537, TA98, TA100 and TA102 (Experiment 1) and for TA1535, TA98 and TA102 (Experiment 2)
- 156.25, 312.5, 625, 1250 and 2500 µg/plate, for TA1535 (Experiment 1)
- 156.25, 312.5, 625, 1250, 2500 and 5000 µg/plate, for TA1537 and TA100 (Experiment 2)
- 1250, 1875, 2500, 3750 and 5000 µg/plate, for TA1537 (Experiment 3)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol; Batch no.: V8M 084248 M (Carlo Erba, 27106 Val de Reuil, France)
- Justification for choice of solvent/vehicle: The test substance was freely soluble in the vehicle
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Anthraminev(2AM)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Anthramine (2AM)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation; experiment 1); pre-incubation (experiment 2)

EXPERIMENTAL PERFORMANCE:
In two independent experiments, five dose-levels of CUMYL PEROXYNEODECANOATE (three plates/dose-level) were tested on each strain, with and without S9 mix. In a complementary experiment, five dose-levels of the test substance (three plates/dose-level) were tested on the TA 1537 strain, with S9 mix.
Direct plate incorporation method (preliminary toxicity test, both experiments without S9 mix, first and third experiment with S9 mix):
- 0.05 mL test substance solution
- 0.5 mL S9 mix
- 0.1 mL bacterial suspension
- mixed with 2 mL of overlay agar (containing traces of relevant amino acid and biotin and maintained at 45 °C)
After rapid homogenization, mixture was overlaid onto Petri plate containing minimum medium.

Preincubation method (second experiment with S9):
- 0.05 mL test substance solution
- 0.5 mL S9 mix
- 0.1 mL bacterial suspension
- mixed with agar after 60 minutes incubation time at 37 °C

After 48 to 72 hours of incubation at 37 °C revertants were scored with an automatic counter

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Preliminary toxicity test: Six dose levels (one plate/dose-level) were tested in the TA98, TA100 and TA102 strains with and without S9 mix. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn
Evaluation criteria:
A reproducible two-fold increase in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained. See Table 1 in box 'Any other information on materials & methods incl. tables' for historical data.

Acceptance criteria:
This study is considered valid if the following criteria are fully met:
- the number of revertants in the vehicle controls is consistent with our historical data (appendix 2)
- the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with our historical data.
Statistics:
not applicable
Key result
Species / strain:
other: TA1535, TA100, TA102, TA 1537, TA98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
EXPERIMENTS WITHOUT S9 MIX:
In the first experiment, a slight to moderate toxicity was induced in the TA1535 and TA98 strains at dose-level ≥1250 µg/plate. In the TA100 strain, a slight thinning of the bacterial lawn was noted at 5000 µg/plate.
In the second experiment, except for a slight toxicity observed in the TA1537 at 2500 µg/plate, no noteworthy toxicity was induced.
In the first experiment, a 2-fold increase in the number of revertants was noted at 312.5 µg/plate in the TA1537 strain. However, this slight increase was not considered as biologically relevant since it was neither induced at higher dose-level nor reproduced in the second experiment. No noteworthy increase in the number of revertant was observed in the remaining tester strains.

EXPERIMENTS WITH S9 MIX:
No toxicity was noted towards all the strains used, in the first experiment.
In the second experiment (preincubation), using the preincubation method, up to 2.8-fold increase in the number of revertants was noted with this tester strain, without any clear evidence of a dose relationship.
Even though the individual and the mean values obtained in these two experiments remained clearly within the vehicle control historical range (3-25), in order to check the reliability of the increase in revertants mainly noted in the first experiment, it was decided to perform a third experiment, using a closer range of dose-levels.
In this third experiment, no noteworthy increase in the number of revertants was induced.
Therefore, the more than two-fold increases previously noted were considered as sporadic and were attributed to the low vehicle control values.
The test substance did not induce any noteworthy increase in the number of revertants, in the remaining tester strains.

For individual results see Table 2 in 'Any other information on results incl. tables'.

Table 2: First experiment without S9 mix

strains

doses* (µg/plate)

T

E

revertants per plate

mean

SD

ratio

TA 1535

0

0

0

8

7

5

7

2

-

156.25

0

0

9

9

3

7

3

1.05

312.5

0

0

15

6

7

9

5

1.40

625

0

0

9

6

4

6

3

0.95

1250

1

1

9

7

10

9

2

1.30

2500

2

1

6

2

3

4

2

0.55

NaN3(l)

-

1

372

408

357

379

26

56.85

TA 1537

0

0

0

7

3

3

4

2

-

312.5

0

0

9

9

8

9

1

2.00

625

0

1

3

9

5

6

3

1.31

1250

0

1

6

4

8

6

2

1.38

2500

0

1

9

8

8

8

1

1.92

5000

0

2

8

12

2

7

5

1.69

9AA(50)

-

-

204

166

183

184

19

42.54

TA 98

0

0

0

22

15

18

18

4

-

312.5

0

0

10

17

16

14

4

0.78

625

0

1

16

23

16

18

4

1.00

1250

1

1

20

14

17

17

3

0.93

2500

1

1

21

27

13

20

7

1.11

5000

2

2

22

9

11

14

7

0.76

2NF(0.5)

-

-

162

160

182

168

12

9.16

TA 100

0

0

0

52

70

73

65

11

-

312.5

0

0

70

59

55

61

8

0.94

625

0

1

61

70

59

63

6

0.97

1250

0

1

64

44

42

50

12

0.77

2500

0

1

68

47

61

59

11

0.90

5000

1

1

54

70

69

64

9

0.99

NaN3(l)

-

-

278

237

296

270

30

4.16

TA 102

0

0

0

145

119

131

132

13

-

312.5

0

0

99

89

79

89

10

0.68

625

0

1

138

132

143

138

6

1.05

1250

0

1

146

99

80

108

34

0.82

2500

0

1

145

165

119

143

23

1.09

5000

0

2

110

107

81

99

16

0.75

MMC(0.5)

-

-

850

663

735

749

94

5.69

0: vehicle control (ethanol)

*: doses expressed as active material

T: toxicity

E: emulsion

ratio: (number of revertants with the test substance)/(number of revertants with vehicle)

Table 3: Second experiment without S9 mix

strains

doses* (µg/plate)

T

E

revertants per plate

mean

SD

ratio

TA 1535

0

0

0

6

12

7

8

3

-

78.125

0

0

8

8

12

9

2

1.12

156.25

0

0

10

8

9

9

1

1.08

312.5

0

0

8

10

3

7

4

0.84

625

0

1

11

7

13

10

3

1.24

1250

0

1

4

9

13

9

5

1.04

2500

0

1

18

11

8

12

5

1.48

NaN3(l)

-

-

420

372

344

379

38

45.44

TA 1537

0

0

0

9

6

2

6

4

-

78.125

0

0

8

8

3

6

3

1.12

156.25

0

0

2

5

4

4

2

0.65

312.5

0

0

6

4

3

4

2

0.76

625

0

1

6

7

11

8

3

1.41

1250

0

1

7

2

11

7

5

1.18

2500

1

1

7

7

6

7

1

1.18

9AA(50)

-

-

173

217

181

190

23

33.59

TA 98

0

0

0

31

27

23

27

4

-

78.125

0

0

19

26

18

21

4

0.78

156.25

0

0

34

28

19

27

8

1.00

312.5

0

0

29

30

15

25

8

0.91

625

0

1

24

26

22

24

2

0.89

1250

0

1

31

25

19

25

6

0.93

2500

0

1

27

26

28

27

1

1.00

2NF(0.5)

-

-

147

177

175

166

17

6.16

TA 100

0

0

0

72

76

56

68

11

-

78.125

0

0

55

64

54

58

6

0.85

156.25

0

0

67

61

68

65

4

0.96

312.5

0

0

64

60

56

60

4

0.88

625

0

1

76

61

54

64

11

0.94

1250

0

1

70

66

67

68

2

1.00

2500

0

1

100

68

90

86

16

1.26

NaN3(l)

-

-

473

461

414

449

31

6.61

TA 102

0

0

0

198

189

197

195

5

-

78.125

0

0

123

111

100

111

12

0.57

156.25

0

0

172

194

131

166

32

0.87

312.5

0

0

154

114

107

125

25

0.64

625

0

1

192

180

197

190

9

0.97

1250

0

1

176

131

126

144

28

0.74

2500

0

1

187

162

149

166

19

0.85

MMC(0.5)

-

-

990

1079

1001

1023

49

5.26

Table 4: First experiment with S9 mix

strains

doses* (µg/plate)

T

E

revertants per plate

mean

SD

ratio

TA 1535

0

0

0

15

8

24

16

8

-

156.25

0

0

16

16

15

16

1

1.00

312.5

0

0

20

23

16

20

4

1.26

625

0

1

13

12

19

15

4

0.94

1250

0

1

20

16

19

18

2

1.17

2500

0

1

27

19

25

24

4

1.51

2AM(2)

-

-

138

143

103

128

22

8.17

TA 1537

0

0

0

5

7

5

6

1

-

312.5

0

0

11

6

2

6

5

1.12

625

0

0

6

12

7

8

3

1.47

1250

0

1

9

13

6

9

4

1.65

2500

0

1

16

13

10

13

3

2.29

5000

0

2

17

13

12

14

3

2.47

2AM(2)

-

-

62

66

70

66

4

11.65

TA 98

0

0

0

22

36

28

29

7

-

312.5

0

0

17

7

21

15

7

0.52

625

0

0

19

26

21

22

4

0.77

1250

0

1

26

16

23

22

5

0.76

2500

0

1

17

23

15

18

4

0.64

5000

0

1

27

24

21

24

3

0.84

2AM(2)

-

-

397

386

371

385

13

13.42

TA 100

0

0

0

87

69

83

80

9

-

312.5

0

0

99

85

105

96

10

1.21

625

0

0

100

94

108

101

7

1.26

1250

0

1

152

128

114

131

19

1.65

2500

0

1

141

154

135

143

10

1.80

5000

0

2

172

166

131

156

22

1.96

2AM(2)

-

-

936

987

897

940

45

11.80

TA 102

0

0

0

194

197

177

189

11

-

312.5

0

0

177

116

111

135

37

0.71

625

0

0

169

191

201

187

16

0.99

1250

0

1

176

136

133

148

24

0.78

2500

0

1

223

152

216

197

39

1.04

5000

0

1

223

184

154

187

35

0.99

2AM(2)

-

-

821

700

719

747

65

3.94

Table 5: Second experiment with S9 mix: Preincubation method

strains

doses* (µg/plate)

T

E

revertants per plate

mean

SD

ratio

TA 1535

0

0

0

13

6

12

10

4

-

312.5

0

0

13

9

8

10

3

0.97

625

0

0

8

15

17

13

5

1.29

1250

0

1

10

13

12

12

2

1.13

2500

0

1

14

16

13

14

2

1.39

5000

0

2

16

17

13

15

2

1.48

2AM(2)

-

-

81

89

90

87

5

8.39

TA 1537

0

0

0

3

5

2

3

2

-

156.25

0

0

5

13

10

9

4

2.80

312.5

0

0

14

2

2

6

7

1.80

625

1

0

5

11

7

8

3

2.30

1250

1

0

11

6

11

9

3

2.80

2500

2

1

11

11

6

9

3

2.80

5000

2

1

8

4

5

6

2

1.70

2AM(2)

-

-

63

49

63

58

8

17.50

TA 98

0

0

0

25

20

20

22

3

-

312.5

0

0

17

23

23

21

3

0.97

625

0

0

20

17

25

21

4

0.95

1250

0

0

25

23

26

25

2

1.14

2500

0

1

21

21

23

22

1

1.00

5000

0

2

35

32

31

33

2

1.51

2AM(2)

-

-

489

399

465

451

47

20.82

TA 100

0

0

0

118

89

101

103

15

-

156.25

0

0

100

103

95

99

4

0.97

312.5

0

0

90

115

95

100

13

0.97

625

0

0

113

103

82

99

16

0.97

1250

0

1

94

82

83

86

7

0.84

2500

1

1

113

111

92

105

12

1.03

5000

1

2

127

120

123

123

4

1.20

2AM(2)

-

-

689

609

558

619

66

6.03

TA 102

0

0

0

205

253

196

218

31

-

312.5

0

0

187

135

175

166

27

0.76

625

0

0

227

187

209

208

20

0.95

1250

0

0

201

216

176

198

20

0.91

2500

0

1

277

319

309

302

22

1.38

5000

0

2

328

243

219

263

57

1.21

2AM(2)

-

-

1012

741

740

831

157

3.81

Conclusions:
In conclusion, the test substance does not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium in the presence or absence of mammalian metabolic activation.
Executive summary:

In a reverse gene mutation assay in bacteria (OECD 471) strains of S. typhimurium (TA1537, TA1535, TA102, TA100 and TA98) were exposed to Cumyl Peroxyneodecanoate (1 -Methyl-1 -phenylethyl peroxyneodecanoate; 70% purity in isododecane) in ethanol at concentrations of 78.125, 156.25, 312.5, 625, 1250, 2500, 3750 and 5000 µg/ plate in the presence and absence of metabolic activation. The positive controls did induce the appropriate response in the corresponding strains. There was no evidence of induced mutant colonies over background.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003-01-14 to 2003-07-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted 21st July 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Supplier: ATOFINA
- Chemical Name: Alpha-CUMYL PEROXYNEODECANOATE
- Batch number: 0210840414
- Description: colourless liquid
- Container: one plastic flask
- Storage conditions: at -20 °C and protected from light
- Purity: 75% in isododecane

The test item was dissolved in the vehicle at a concentration of 612.9 mg/mL for the first experiment and 306.45 mg/mL for the second experiment.
The preparations were made immediately before use.
Species / strain / cell type:
lymphocytes: human lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Cells used: Human lymphocytes
- Modal number of chromosomes: 46
- Cell cycle time: 12 - 14
- Human lymphocytes were prepared from whole blood samples obtained from two healthy donors and collected into heparinised sterile tubes.

MEDIA USED
RPMI 1640 medium containing 20% fetal calf serum, L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 μg/mL) and phytohaemagglutinin
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Rat liver post-mitochondrial fraction (S9)
Test concentrations with justification for top dose:
The highest dose-level for treatment in the first experiment was selected on the basis of pH, osmolality and solubility. For selection of the dose-levels for the second experiment, toxicity indicated by the reduction of mitotic index (MI) in the first experiment, if any, was also taken into account.
- Experiment I (with and without S9): 0.16, 0.31, 0.63, 1.25, 2.5, 5, and 10 mM
- Experiment II (with S9): 0.16, 0.31, 0.63, 1.25, 2.5 and 5 mM
- Experiment II (without S9): 0.04, 0.08, 0.16, 0.31, 0.63 and 1.25 mM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol, batch no. V8M084248 M (Carlo Erba, 27106 Val de Reuil, France)
Untreated negative controls:
yes
Remarks:
distilled water
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9: 3 µg / mL ( 3h) or 0.2 µg / mL (continuous experiment)
Untreated negative controls:
yes
Remarks:
distilled water
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9: 12.5, 25 or 50 µg / mL
Details on test system and experimental conditions:
Treatment
In two independent experiments, using duplicate cultures, the cells were tested, with and without S9 mix, with:
- at least five dose-levels of the test item,
- the vehicle control,
- the appropriate positive control.
To prepare each culture, 0.5 mL of heparinised whole blood was added to 5 mL of RPMI 1640 medium containing 20% fetal calf serum, L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 μg/mL) and phytohaemagglutinin (PHA: a mitogen to stimulate the lymphocytes to divide). The cultures were then placed at 37°C for 48 hours.

In the first experiment, lymphocyte cultures were then exposed to the test or control items, both in the absence and presence of S9 mix, for 3 hours then rinsed. One and a half hour before harvest, each culture was treated with a colcemid solution (10 μg/mL) to block cells at the metaphase-stage of mitosis. Harvest time was 20 hours from the beginning of treatment, corresponding to approximately 1.5 normal cell cycles.

The second experiment was performed as follows:
• without S9 mix, cells were exposed continuously to the test or control items, until harvest,
• with S9 mix, cells were exposed to the test or control items for 3 hours and then rinsed.
One and a half hour before harvest, each culture was treated with a colcemid solution (10 μg/mL) to block cells at the metaphase-stage of mitosis. Harvest times were 20 hours and 44 hours from the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later.

Preparation of microscope slides
After hypotonic treatment (KCl 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa. All the slides were coded, so that the scorer is unaware of the treatment group of the slide under evaluation ("blind" scoring).

Determination of cytotoxicity
The cytotoxicity of the test item was evaluated using the mitotic index (number of cells in mitosis/1000 cells examined), which indicates whether a item induces mitotic inhibition. Mitotic index was determined without blind scoring. Analysis of 200 metaphases/dose-level (with 44 to 46 chromosomes) was made, with 100 metaphases/culture whenever possible. Only 50 metaphases/culture were analysed when at least 10% cells with structural chromosome aberrations were observed. All metaphase analyses were performed blind. The following structural aberrations were recorded for each metaphase (c, d): gaps, chromatid and chromosome breaks and exchanges, and others (multiple aberrations and pulverizations). In addition, the following numerical aberrations were recorded when encountered: polyploidy and endoreduplication. The metaphase analysis of the slides was performed at Microptic, cytogenetic services (2 Langland Close Mumbles, Swansea SA3 4LY, UK), in compliance with GLP, and the Principal Investigator was Natalie Danford.
Evaluation criteria:
A reproducible and statistically significant increase in the frequency of cells with structural chromosome aberrations for at least one of the dose-levels and one of the two harvest times was considered as a positive result. Reference to historical data or other considerations of biological relevance, was also taken into account in the evaluation of the findings.
Statistics:
For each test and for each harvest time, the frequency of cells with structural chromosome aberrations (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. If necessary, the comparison was performed using the χ2 test, in which p= 0.05 was used as the lowest level of significance.
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: pH was approximately 7.7 (7.4 for the vehicle control) and the osmolality equal to 313 mOsm/kg H2O (377 for the vehicle control).
- Other confounding effects: No emulsion was observed in the culture medium at the end of the treatment period.

Experiments without S9 mix:

Cytotoxicity:
Following the 3-hour treatment, a moderate to marked decrease in mitotic index (56-81% decrease) was noted at dose-levels ≥ 1.25 mM. Following the 20-hour treatment, a moderate decrease in mitotic index (41% decrease) was noted at 1.25 mM. Following the 44-hour treatment, a slight to marked decrease in mitotic index (31-70% decrease) was noted at dose-levels ≥ 0.63 mM.

Metaphase analysis:
The dose-levels selected for metaphase analysis were as follows:
- 0.31, 0.63 and 1.25 mM for the 3-hour and the 20-hour treatments, the latter inducing 56 and 41% decreases in mitotic index, respectively,
- 1.25 mM, for the 44-hour treatment, this dose-level inducing 70% decrease in mitotic index.
No significant increase in the frequency of cells with structural chromosomal aberrations was noted after 3, 20 as well as 44-hour treatments.


Experiments with S9 mix:

Cytotoxicity:
A slight to marked decrease in mitotic index (29-78% decrease) was noted at dose-levels ≥ 0.63 mM at the 20-hour harvest time.
At the 44-hour harvest time, a moderate to strong decrease in mitotic index was noted at dose-levels ≥ 1.25 mM (59-89% decrease).

Metaphase analysis:
The dose-levels selected for metaphase analysis were as follows:
- 1.25, 2.5 and 5 mM, for the 20-hour harvest time in the first experiment, the latter inducing 59% decrease in mitotic index,
- 0.31, 0.63 and 1.25 mM, for the 20-hour harvest time in the second experiment, the latter inducing 68% decrease in mitotic index,
- 1.25 mM, for the 44-hour harvest time, this dose-level inducing 59% decrease in mitotic index.
No significant increase in the frequency of cells with structural chromosomal aberrations was noted in both experiments and at both harvest times. The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid. The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid.

Table 1: Experiment 1 without S9 mix: chromosome aberration (3 -hour treatment, 20 -hour harvest)

Doses mM Slide Nb. Nb. Of cells scored NA Structural chromosome aberrations
(type and number)
Cells with structural chromosome aberrations
G Chromatid Chromosome MA PU Total
+G
Total
-G
Nb.
+G
% Nb.
-G
%
D Exch D Exch
0 14M 100 0 0 1 0 0 0 0 0 5 3 1 2.0 1 1.5
23F 100 1 2 0 0 1 1 0 0 3 2
0.31 22M 100 1 2 1 0 2 2 0 0 9 7 6 4.0 4 3.0
6F 100 0 0 1 1 0 0 0 0 2 2
0.63 5M 100 0 2 0 0 0 0 0 0 6 2 2 3.0 0 1.0
15F 100 1 2 2 0 0 0 0 0 4 2
1.25 39M 100 2 3 1 0 1 1 0 0 10 5 6 5.0 3 2.5
12F 100 0 2 2 0 0 0 0 0 4 2
MMC 9M 50 0 4 18 11 3 0 0 0 89 78 21 52.0 19 48.0
***
3µg/mL 18F 50 0 7 28 14 4 0 0 0 31 29

Table 2: Second experiment without S9 mix: chromosome aberration (20 -hour treatment, 20 -hour harvest)

Doses mM Slide Nb. Nb. of cells scored NA Structural chromosome aberrations
(type and number)
Cells with structural chromosome aberrations
G Chromatid Chromosome MA PU Total
+G
Total
-G
Nb.
+G
% Nb.
-G
%
D Exch D Exch
0 48M 100 0 2 2 0 0 0 0 0 5 3 4 2.5 1 1.5
57F 100 2 0 0 0 1 0 0 0 1 2
0.31 51M 100 0 1 0 0 0 0 0 0 3 1 1 1.5 4 0.5
75F 100 0 1 1 0 0 0 0 0 2 2
0.63 64M 100 1 1 1 0 0 0 0 0 6 1 2 3.0 0 0.5
44F 100 0 4 0 0 0 0 0 0 4 2
1.25 60M 100 0 0 0 0 0 0 0 0 3 2 0 1.5 3 1.0
71F 100 1 1 2 0 0 0 0 0 3 2
MMC 54M 50 0 3 5 4 1 0 0 0 30 23 10 24.0 19 21.0
***
3 µg/mL 49F 50 0 4 6 5 2 0 0 0 14 29

Table 3: Experiment 2 without S9 mix: chromosome aberration (44 -hour treatment, 44 -hour harvest)

Doses mM Slide Nb. Nb. of cells scored NA Structural chromosome aberrations
(type and number)
Cells with structural chromosome aberrations
G Chromatid Chromosome MA PU Total
+G
Total
-G
Nb.
+G
% Nb.
-G
%
D Exch D Exch
0 84M 100 0 2 0 0 2 0 0 0 4 2 4 2.0 2 1.0
97F 100 1 0 0 0 0 0 0 0 0 0
Jan 25 81M 52 0 1 2 0 0 0 0 0 15 11 3 5.5 2 3.5
87F 148 3 3 2 0 7 0 0 0 8 5

Table 4: Experiment 1 with S9 mix: chromosome aberration (3 -hour treatment, 20 -hour harvest)

Doses mM Slide Nb. Nb. of cells scored NA Structural chromosome aberrations
(type and number)
Cells with structural chromosome aberrations
G Chromatid Chromosome MA PU Total
+G
Total
-G
Nb.
+G
% Nb.
-G
%
D Exch D Exch
0 32M 100 0 1 1 0 0 0 0 0 5 3 2 2.5 1 1.5
8F 100 0 1 0 0 2 0 0 0 3 2
1.25 2M 100 1 4 0 0 1 0 0 0 9 2 5 4.5 1 1.0
33F 100 1 3 0 0 0 1 0 0 4 1
2.5 38; 100 2 3 1 0 1 1 0 0 13 9 6 5.5 3 3.5
26F 100 1 1 3 0 3 0 0 0 5 4
5 16M 100 0 7 0 0 0 0 0 0 10 1 7 5.0 0 0.5
4F 100 3 2 0 0 1 0 0 0 3 1
CPA 27M 50 0 6 26 5 1 0 0 0 91 78 22 52.0 21 48.0
***
50 µg/mL 17F 50 0 7 35 6 4 0 1 0 30 27

Table 5: Experiment 2 with S9 mix: chromosome aberration (4 -hour treatment 20 -hour harvest)

Doses mM Slide Nb. Nb. Of cells scored NA Structural chromosome aberrations
(type and number)
Cells with structural chromosome aberrations
Chromatid Chromosome MA PU Total
+G
Total
-G
Nb.
+G
% Nb.
-G
%
D Exch D Exch
0 43M 100 0 1 0 0 0 0 0 5 4 1 2.5 1 2.0
53F 100 0 2 0 1 0 0 0 4 3
0.31 52M 100 0 0 0 0 0 0 0 3 1 0 1.5 0 0.5
69F 100 2 1 0 0 0 0 0 3 1
0.63 66M 100 1 1 0 0 0 0 0 5 4 1 2.0 1 1.5
47F 100 0 1 0 1 1 0 0 3 2
Jan 25 56M 100 0 1 0 0 0 0 0 6 4 3 3.0 1 2.0
72F 100 0 1 0 1 1 0 0 3 3
CPA 73M 50 0 10 3 3 0 0 0 38 31 16 28.0 13 23.0
***
12.5 µg/mL 45F 50 0 12 2 1 0 0 0 12 10

Table 6: Experiment 2 with S9 mix: chomosome aberration (3 -hour treatment 44 -hour harvest)

Doses mM Slide Nb. Nb. Of cells scored NA Structural chromosome aberrations
(type and number)
Cells with structural chromosome aberrations
Chromatid Chromosome MA PU Total
+G
Total
-G
Nb.
+G
% Nb.
-G
%
D Exch D Exch
0 77M 100 0 1 1 0 0 0 0 3 1 2 1.5 1 0.5
91F 100 0 0 0 0 0 0 0 1 0
1.25 83M 100 0 2 1 0 0 0 0 5 2 3 2.5 1 1.0
94F 100 1 1 1 0 0 0 0 2 1

NA: numerical aberrations, G: gap, D: deletion, Exch: exchange, MA: multiple aberrations, PU: pulverization.

M: male

F: female

0: vehicle control (DMSO)

MMC: mitomycin C

Statistical analysis: Chi-Quadrat test ***: p < 0.001 (performed only for cells with structural aberrations excluding gaps)

Nb.: number

(1): expressed as active

Conclusions:
In this study, the test substance did not induce chromosome aberrations in the performed experiments with or without metabolic activation. Therefore, the test item is not considered to be clastogenic in this test system.
Executive summary:

In an in-vitro mammalian chromosome aberration test conducted in accordance with OECD 473, the potential of the test item (75% in isododecane) to induce chromosome aberrations in cultured human lymphocytes was evaluated in the presence and absence of a liver metabolizing system (S9). In the first experiment, lymphocytes were exposed to 0.16, 0.31, 0.63, 1.25, 2.5, 5, and 10 mM with and without S9 mix for 3 hours, then rinsed. Cells were harvested 20 hours after beginning of treatment. For the second experiment, the cells were treated with 0.16, 0.31, 0.63, 1.25, 2.5 and 5 mM with S9 mix for 3 hours, then rinsed and with 0.04, 0.08, 0.16, 0.31, 0.63 and 1.25 mM without S9 mix until harvest. Cells were harvested 20 and 44 hours after the beginning of treatment. For all tested treatment groups, no dose-response relationship could be observed. The positive controls did induce the appropriate response. There was no significant increase in the frequency of cells with structural chromosomal aberrations in both experiments and at both harvest times. Based on these results, the test item did not induce chromosome aberrations in cultured human lymphocytes. 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-03-24 to 2017-08-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
TEST MATERIAL
- Name: 1-methyl-1-phenylethyl peroxyneodecanoate
- Product name: Peroxan CND
- CAS No.: 26748-47-0
- Physical state: fluid
- Colour: colourless/yellowish
- Density: 0.962 g/cm³
- Active component: 69.6% peroxide

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: ; Batch no.: 1207870-01
- Purity test date: 2017-02-21


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ≤ -20 °C

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: A solubility test was performed with different solvents and vehicles. Based on the results of the solubility test THF was most suitable. The test item was dissolved in THF and diluted prior to treatment. The different standard solutions were added to cell culture medium (MEM + 0% FBS 4 h treatment) with a final concentration of 0.5% (v/v). The solvent was compatible with the survival of the cells and the S9 activity. The pH-value detected with the test item was within the physiological range (pH 7.0 ± 0.4). Osmolality of the highest test item concentration in experiment 1 (250 µg/mL) was 373 mOsmol/kg.
Target gene:
HPRT (hypoxanthine-guanine phosphoribosyl transferase) locus of V79 cells
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELL SOURCE
- Source of cells: Eurofins BioPharma Product Testing Munich GmbH
- Suitability of cells: V79 cells in vitro have been widely used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens. These cells are characterized by their high proliferation rate.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Complete Culture Medium (MEM medium supplemented with 10% fetal bovine serum (FBS), 100 U/100 µg/mL penicillin/streptomycin, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B)
Treatment Medium (MEM medium supplemented with 100 U/100 µg/mL penicillin/streptomycin, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B)
Selective Medium (MEM medium supplemented with 10% fetal bovine serum (FBS), 100 U/100 µg/mL penicillin/streptomycin, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B, 11 µg/mL thioguanine (TG))
- Periodically checked for Mycoplasma contamination: yes (each cell batch)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Pre-experiment: 25, 50, 100, 250, 500, 1000, 1500 and 2000 µg/mL

The selection of the concentrations used in the main experiments was based on data from the pre-experiments according to the OECD guideline 476.

Experiment 1:
without metabolic activation: 0.5, 1, 2, 5, 10, 30, 50, 100 and 250 µg/mL
with metabolic activation: 1, 2, 5, 10, 20, 30, 40, 50, 100 and 250 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tetrahydrofuran
- Justification for choice of solvent/vehicle: A solubility test was performed with different solvents and vehicles. Based on the results, tetrahydrofuran was most suitable.
Untreated negative controls:
yes
Remarks:
treatment medium, duplicate cultures
Negative solvent / vehicle controls:
yes
Remarks:
tetrahydrofuran, Applichem Lot No. I864407
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation; final concentration: 300 µg/mL
Untreated negative controls:
yes
Remarks:
treatment medium, duplicate cultures
Negative solvent / vehicle controls:
yes
Remarks:
tetrahydrofuran, Applichem Lot No. I864407
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation; final concentrations: 1.0 and 1.5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 5 x 10^6 cells per negative, solvent and positive controls were seeded in complete culture medium (with and without metabolic activation) and in total 10 x 10^6 cells for each test item concentration.

DURATION
- Preincubation period: 24 hours
- Exposure duration: 4 h
- Expression time (cells in growth medium): 6-7 days
- Selection time (if incubation with a selection agent): 7-9 days

SELECTION AGENT (mutation assays): 6-thioguanine

STAIN (for cytogenetic assays): Giemsa

NUMBER OF CELLS EVALUATED: approx. 200

DETERMINATION OF CYTOTOXICITY
- Method: Relative survival, based on the cloning efficiency
Evaluation criteria:
A test chemical is considered to be clearly negative if, in all experimental conditions examined
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend-test
- all results are inside the distribution of the historical negative control data.

A test chemical is considered to be clearly positive if, in any of the experimental conditions examined
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control, and
- the increase is concentration-related when evaluated with an appropriate trend test, and
- any of the results are outside the distribution of the historical negative control data
- if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.

According to the OECD guideline, the biological relevance is considered first for the interpretation of results.
Statistics:
The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls. Mutant frequencies of the negative/solvent controls were used as reference.
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:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test item was noted at concentrations of 100 µg/mL and higher (with metabolic activation).

RANGE-FINDING/SCREENING STUDIES
The selection of the concentrations used in the main experiments was based on data from the pre-experiments according to the OECD guideline 476.
In experiment 1 250 µg/mL (without metabolic activation) and 100 µg/mL (with metabolic activation) were selected as the highest concentrations due to cytotoxic effects and precipitation, respectively. Experiment 1 with and without metabolic activation was performed as a 4 h short-term exposure assay.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The positive controls, DMBA (1 and 1.5 µg/mL) and EMS (300 µg/mL) showed statistically significant increases in mutant frequency, thereby demonstrating both the sensitivity and validity of the test systems.
- Negative (solvent/vehicle) historical control data: The mean mutant value of the negative and solvent controls fall within the historical data range of the test facility and the cloning efficiencies of the negative and solvent controls are > 50%.

ADDITIONAL INFORMATION ON CYTOTOXICITY
A biologically relevant growth inhibition (reduction of relative survival below 70%) was observed after the treatment with the test item in experiment 1 without metabolic activation.
In experiment 1 without metabolic activation the relative survival was 17% for the highest concentration (250 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 100 µg/mL due to precipitation with a relative survival of 88%.

For individual results see Table 2 to 5 in box 'Any other information on results incl. tables'.

Table 2: Experiment 1 - Toxicity, without metabolic activation

Dose group

Concentration

µg/mL

Number of cells at the

Number of colonies per flask

CE [%]

Adjusted CE [%]

Relative Survival (RS) [%]

beginning of treatment

end of treatment

I

II

mean

NC1

0

10,000,000

10,931,000

147

149

148

74

81

122

NC2

10,000,000

11,509,000

159

139

149

75

86

129

S1

0

10,000,000

8,942,000

163

166

165

82

74

100

S2

10,000,000

8,619,000

136

140

138

69

59

1

0.5

20,000,000

17,374,000

151

133

142

71

62

93

2

1

20,000,000

16,592,000

166

172

169

85

70

105

3

2

20,000,000

15,402,000

144

143

144

72

55

83

4

5

20,000,000

16,558,000

127

127

127

64

53

79

5

10

20,000,000

12,512,000

128

137

133

66

41

62

6

30

20,000,000

13,736,000

141

169

155

78

53

80

7

50

20,000,000

15,062,000

161

155

158

79

59

89

8

100

20,000,000

12,444,000

163

169

166

83

52

78

9

250

20,000,000

4,148,000

102

116

109

55

11

17

PC1

300

10,000,000

11,220,000

171

181

176

88

99

148

PC2

300

10,000,000

10,693,000

153

173

163

82

87

131

PC: positive control (ethylmethanesulfonate)

NC: negative control

S: solvent control (tetrahydrofuran)

CE: cloning efficiency

Table 3: Experiment 1 - Mutagenicity, without metabolic activation

Dose group

Concentration

µg/mL

CE in non-selective medium

CE in selective medium

Mutant frequency per 106 cells

Number of colonies per flask

CE [%]

Number of colonies per flask

I

II

mean

I

II

III

IV

V

mean

SD

NC1

0

154

146

150

75

9

15

8

13

14

11.8

2.8

39.3

NC2

140

126

133

67

8

7

10

4

4

6.6

2.3

24.8

S1

0

173

157

165

83

7

9

3

11

6

7.2

2.7

21.8

S2

133

143

138

69

10

9

8

8

9

8.8

0.7

31.9

1

0.5

143

174

159

79

8

11

8

8

9

8.8

1.2

27.8

2

1

160

157

159

79

12

11

13

14

16

13.2

1.7

41.6

3

2

150

157

154

77

9

9

9

14

15

11.2

1.7

36.5

4

5

176

148

162

81

11

9

8

16

15

11.8

3.2

36.4

5

10

120

150

135

68

3

8

8

5

4

5.6

2.1

20.7

6

30

120

122

121

61

8

13

9

10

16

11.2

2.9

46.3

7

50

124

132

128

64

8

9

10

5

4

7.2

2.3

28.1

8

100

148

142

145

73

12

8

9

4

8

8.2

2.6

28.3

9

250

150

145

148

74

4

10

10

9

10

8.6

2.3

29.2

PC1

300 µg/mL

131

149

140

70

62

72

80

74

86

74.8

8.1

267.1

PC2

300 µg/mL

160

135

148

74

76

68

68

80

77

73.8

4.9

250.2

PC: positive control (ethylmethanesulfonate)

NC: negative control

S: solvent control (tetrahydrofuran)

CE: cloning efficiency

SD: standard deviation

Table 4: Experiment 1 - Toxicity, with metabolic activation

Dose group

Concentration µg/mL

Number of cells at the

Number of colonies per flask

CE [%]

Adjusted CE [%]

Relative survival (RS) [%]

beginning of treatment

end of treatment

I

II

mean

NC1

0

10,000,00

14,671,000

175

192

184

92

135

124

NC2

10,000,00

14,195,000

195

185

190

95

135

125

S1

0

10,000,00

12,546,000

163

156

160

80

100

100

S2

10,000,00

12,240,000

204

176

190

95

116

1

1

10,000,00

12,376,000

198

178

188

94

116

108

2

2

10,000,00

12,546,000

195

234

215

107

135

124

3

5

10,000,00

13,855,000

175

159

167

84

116

107

4

10

10,000,00

12,376,000

172

192

182

91

113

104

5

20

10,000,00

11,883,000

208

196

202

101

120

111

6

30

10,000,00

11,220,000

189

174

182

91

102

94

7

40

10,000,00

12,733,000

153

164

159

79

101

93

8

50

10,000,00

12,053,000

208

205

207

103

124

115

9 P

100

10,000,00

11,016,000

183

162

173

86

95

88

10 P

250

10,000,00

10,472,000

187

168

178

89

93

86

PC 1

1

10,000,00

13,311,000

153

162

158

79

105

97

PC 2

1.5

10,000,00

14,246,000

101

104

103

51

73

67

PC: positive control (ethylmethanesulfonate)

NC: negative control

S: solvent control (tetrahydrofuran)

CE: cloning efficiency

P: precipitation at the end of treatment

SD: standard deviation

Table 5: Experiment 1 - Mutagenicity, with metabolic activation

Dose group

Concentration

µg/mL

CE in non-selective medium

CE in selective medium

Mutant frequency per 106 cells

Number of colonies per flask

CE [%]

Number of colonies per flask

I

II

mean

I

II

III

IV

V

mean

SD

NC1

0

179

166

173

86

16

11

11

16

14.0

14.0

2.4

40.6

NC2

147

160

154

77

11

7

9

11

10.2

10.2

2.0

33.2

S1

0

144

155

150

75

7

10

10

5

7.8

7.8

1.9

26.1

S2

162

154

158

79

7

7

4

4

5.6

5.6

1.4

17.7

4

10

165

160

163

81

5

7

5

7

6.0

6.0

0.9

18.5

5

20

171

137

154

77

10

13

5

16

11.4

11.4

3.7

37.0

6

30

160

180

170

85

8

15

9

13

10.8

10.8

2.7

31.8

7

40

160

172

166

83

21

15

17

10

15.6

15.6

3.6

47.0

8

50

149

170

160

80

14

11

17

12

13.0

13.0

2.3

40.8

9 P

100

153

160

157

78

6

5

11

6

7.8

7.8

2.6

24.9

PC1

1

156

137

147

73

146

153

159

142

151.6

151.6

6.7

517.4

PC2

1.5

152

131

142

71

205

192

208

179

192.8

192.8

12.1

681.3

Conclusions:
Under the experimental conditions reported, the test item 1-Methyl-1-phenylethyl peroxyneodecanoate is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.
Executive summary:

In a mammalian cell HPRT gene mutation assay according to guideline OECD 476, strains of Chinese hamster lung fibroblasts (V79) were exposed to 1 -Methyl-1 -phenylethyl peroxyneodecanoate (69.6% Peroxide) in tetrahydrofuran at concentrations of 0.5, 1, 2, 5, 10, 30, 50, 100 and 250 µg/mL (without metabolic activation) and (with metabolic activation) 1, 2, 5, 10, 20, 30, 40, 50, 100 and 250 µg/mL. The positive controls did induce the appropriate response in the corresponding strains. There was no evidence of induced mutant colonies over background. According to the clear negative results observed in the first experiment (with and without metabolic activation), the second experiment with long time exposure (without metabolic activation) was not considered necessary.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 476 for in vitro mammalian cell transformation.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The target substance 1-Methyl-1-phenylethyl peroxyneodecanoate was tested in an in vitro genotoxicity testing battery as required by Annex VIII of the REACH regulation 1907/2006 (OECD 471, 473 and 476, GLP).

In a bacterial reverse gene mutation test conducted according to OECD 471, the target substance did not induce mutagenicity. In a mammalian cell HPRT mutation assay conducted according to OECD 476, the target substance 1-Methyl-1-phenylethyl peroxyneodecanoate was also tested negative for inducing mutagenic effects. Moreover, the target substance was tested negative in an in vitro cytogenicity study in human lymphocytes conducted according to OECD 473. Based on the lack of mutagenicity/cytogenicity in all in vitro assays, 1-Methyl-1-phenylethyl peroxyneodecanoate is considered to be non-genotoxic.

Justification for classification or non-classification

Based on the lack of mutagenicity/cytogenicity in all conducted in vitro assays with the target substance, 1-Methyl-1-phenylethyl peroxyneodecanoate is considered to be non-genotoxic and no classification is warranted in accordance with CLP Regulation 1272/2008.