Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test: Dimyristylperoxydicarbonate is nonmutagenic in the Ames test with the strains TA 97a, TA 98, TA 100, TA 102 and TA 1535 with and without an external metabolising system up to 1667 μg/plate, which is the limit of solubility.

HPRT Test: The test item Dimyristylperoxydicarbonate was not mutagenic in this in vitro mammalian cell gene mutation test performed with CHO-K1 (Chinese hamster ovary) cells.

Chromosome Aberration Test: The test item is considered to be non-clastogenic in this chromosome aberration test, when tested up to precipitating concentrations.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-09-22 to 2011-10-24
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:
30 May 2008
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 mix
Test concentrations with justification for top dose:
In the first experiment:
21, 62, 185, 556 and 1667 μg per plate without external metabolisation, and
21, 62, 185, 556 and 1667 μg per plate with S9-mix from Aroclor 1254 induced microsomes of rat liver as an external metabolising system.
In the second experiment:
7, 21, 62, 185 and 556 μg per plate without external metabolisation, and
7, 21, 62, 185 and 556 μg per plate with S9-mix from Aroclor 1254 induced microsomes of rat liver as an external metabolising system.
Vehicle / solvent:
Acetone
- Vehicle/solvent used: acetone
- Justification for choice of solvent/vehicle: The test substance was not soluble in water or DMSO. Acetone is a common vehicle for the Ames test.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene diamine (4-NOPD)
Remarks:
TA 97a, without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 100, TA 1535 without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminonthracene (2-AA)
Remarks:
TA 98, TA 100, TA 1535 with S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA 98 without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
TA 97a with S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 1,8-dihydroxyanthraquinone (DHA)
Remarks:
TA 102 with S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: t-butyl-hydroperoxide (tBHPO)
Remarks:
TA 102 without S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation;

DURATION
- Preincubation period: 20 min
- Exposure duration: 2 days

NUMBER OF REPLICATIONS: 3 per dose and positive controls; 6 per vehicle control

DETERMINATION OF CYTOTOXICITY
- Method: reduced or no bacterial background lawn, cloning efficiency
Evaluation criteria:
Means and standard deviations were calculated for the number of mutants in every concentration group.
The criteria for a positive result are:
A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:
• For the strains with a low spontaneous revertant rate i.e. TA98 and TA1535: The 2½ fold of the amount of the spontaneous revertants.
• For the strains with a high spontaneous revertant rate i.e. TA97a, TA100 and TA102: The 1 2/3 fold of the amount of the spontaneous revertants.
These threshold values were derived from the variations in the control samples of our historic data of the Ames test.
Statistics:
NA
Key result
Species / strain:
other: Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
(1667 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: no
- Precipitation: at 1667 µg/plate
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: In the preliminary test and in the main test no toxicity was seen up to 1667 μg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA: yes
Conclusions:
Dimyristylperoxydicarbonate is nonmutagenic in the Ames test with the strains TA 97a, TA 98, TA 100, TA 102 and TA 1535 with and without an external metabolising system up to 1667 μg/plate, which is the limit of solubility.
Executive summary:

The test item Dimyristylperoxydicarbonate was tested for mutagenic activity with the "Salmonella typhimurium Reverse Mutation Test" (Ames Test).

The study was conducted in accordance with the OECD-guideline 471 and the Council Regulation (EC) No 440/2008, Method B.13/14. The test substance was dissolved in acetone.

The following concentrations were tested:

In the first experiment:

21, 62, 185, 556 and 1667 μg per plate without external metabolisation, and

21, 62, 185, 556 and 1667 μg per plate with S9-mix from Aroclor 1254 induced microsomes of rat liver as an external metabolising system.

In the second experiment:

7, 21, 62, 185 and 556 μg per plate without external metabolisation, and

7, 21, 62, 185 and 556 μg per plate with S9-mix from Aroclor 1254 induced microsomes of rat liver as an external metabolising system.

In the first experiment the test was performed according to the "direct plate incorporation method", in the second experiment according to the "preincubation method".

As test system the bacterial strains Salmonella typhimurium TA 97a, TA 98, TA 100, TA 102 and TA 1535 were used.

Negative and positive controls were included.

According to the results obtained in this study, Dimyristylperoxydicarbonate is nonmutagenic in the Ames test with the strains TA 97a, TA 98, TA 100, TA 102 and TA 1535 with and without an external metabolising system up to 1667 μg/plate, which is the limit of solubility.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-11-02 to 2011-11-15
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:
21 July 1997
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
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: In Vitro Mammalian Cell Gene Mutation Test
Target gene:
hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt)
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
Sub-line (KI)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F12 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no
Each batch of frozen cells was purged (GEN 013) of HPRT mutants and was free for mycoplasma infections, tested by Central Agricultural Office National Animal Health Institute Budapest, Hungary
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver
Test concentrations with justification for top dose:
Experiment 1, 5-hour treatment period without S9 mix:
80, 90, 100, 105, 110, 115 and 120 µg/mL
Experiment 1, 5-hour treatment period with S9 mix:
20, 60, 100, 120, 140, 160 and 170 µg/mL
Experiment 2, 20-hour treatment period without S9 mix:
10, 20, 25, 30, 32.5, 35 and 37.5 µg/mL
Experiment 2, 5-hour treatment period with S9 mix:
20, 60, 100, 120, 140, 160 and 170 µg/mL
Vehicle / solvent:
The test item was dissolved in N,N-dimethylformamide and diluted prior to treatment. Ditetradecyl peroxydicarbonate prepared in a concentration of 25 mg/mL with N,N-dimethylformamide (stock solution) at the first step. The necessary amount of ditetradecyl peroxydicarbonate was weighed into a calibrated volumetric flask. A partial volume of N,N-dimethylformamide was added and the formulation was stirred until homogeneity was reached. The formulation was diluted by serial dilutions to obtain the dosing formulations for lower doses. The appropriate amount of these dosing formulations were diluted with Ham's F12 medium or Ham's F12 medium + S9 mix to obtain the test concentrations. All dose formulations were prepared directly prior to the treatment of the cells.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
With S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 and 20 hrs
- Expression time: 19 and 24 hrs
During the phenotypic expression period the cultures were subcultured. Aliquots of 5E05 cells were taken on days 1, 3, and 6, and evaluated on day 8.

SELECTION AGENT: 6-thioguanine (6 TG)

NUMBER OF REPLICATIONS: 1

NUMBER OF CELLS EVALUATED: 200 cells/60-mm dish in 5 mL of F12-10 medium were used for cloning efficiency determinations

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
The test item would have been considered to be mutagenic in this assay if all the following criteria were met:
- The assay is valid.
- The mutant frequency at one or more doses is significantly greater than that of the relevant control.
- Increase of the mutant frequency is reproducible.
- There is a clear dose-response relationship.
The test item would have been considered to have shown no mutagenic activity if no increases were observed which met the criteria listed above.
Statistics:
Statistical analysis was done with SPSS PC+ software for the following data:
- mutant frequency between the negative (solvent) and the test item or positive control item treated groups.

The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity is detected, a one-way analysis of variance was carried out. If the obtained result is positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences.
Where significant heterogeneity is found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there is a positive result, the inter-group comparisons are performed using the Mann-Whitney U-test.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
Sub-line (KI)
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
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: no
- Precipitation: No

RANGE-FINDING/SCREENING STUDIES:
Treatment concentrations for the mutation assay were selected on the basis of the result of a Pre-test on cell toxicity. A dose selection (cytotoxicity assay) was performed. During the cytotoxicity assay, 1-3 day old cultures (more than 50 % confluent) were trypsinised and cell suspensions were prepared in Ham's F12 medium. Cells were seeded into 90 mm petri dishes (tissue culture quality: TC sterile) at 106 cells each and incubated in culture medium. After 24 hours the cells were treated with the suitable concentrations of the test item in absence or in presence (8-9 concentrations) of S9 mix (50 µL/mL) and incubated at 37 °C for 5 hours. After the treatment cells were washed and incubated in fresh Ham's F12 medium for 19 hours. Additional groups of cells were treated for 20 hours without metabolic activation (8 concentrations). 24 hours after the beginning of treatment, the cultures were washed with Ham's F12 medium covered with trypsin-EDTA solution and counted and the cell concentration was adjusted to 40 cells/mL with Ham's F12 medium. For each dose, 5 mL was plated in parallel into 3 sterile dishes (diameter is approx. 60 mm). The dishes were incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air for 5-7 days for colony growing. Colonies were then fixed with methanol, stained with Giemsa and the colonies were counted. Survivals were assessed by comparing the colony forming ability of the treated groups to the negative (solvent) control. Precipitation of the test item in the final culture medium was examined visually at beginning and end of the treatments.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mutation frequencies of the positive and negative control cultures were consistent with the historical control data from the previous studies.

 

 

concentration [µg/mL]

S9 mix

survival to treatment

relative population growth [%] of control

total mutant colonies (from 5 dishes)

Absolute Cloning Efficiency

mutant frequency * 10^-6

survival to treatment

relative population growth [%] of control

total mutant colonies (from 5 dishes)

Absolute Cloning Efficiency

mutant frequency * 10^-6

mean colony number ± SD

% vehicle control

mean colony number ± SD

% vehicle control

Experiment 1 (5 h treatment)

 

 

culture I

culture II

Untreated control

 

-

193.3 ± 3.51

102

102

2

100

2

194 ± 4.36

101

102

3

100

3

Negative control

 

-

190 ± 3.61

100

100

4

99

4.04

193 ± 4.36

100

100

3

98

3.06

Positive control (EMS)

1

-

66 ± 6

35

70

533

70

761.43

73 ± 2.65

38

71

547

70

781.43

Test item

80

-

185 ± 2.65

97

100

4

99

4.04

187.7 ± 4.04

97

98

1

96

1.04

90

-

182.7 ± 2.52

96

94

2

93

2.15

179.7 ± 2.08

93

95

4

93

4.3

100

-

166.3 ± 2.08

88

93

2

92

2.17

164.7 ± 2.08

85

93

2

91

2.2

105

-

134 ± 2.65

71

94

4

93

4.3

133.3 ± 1.53

69

93

5

91

5.49

110

-

99 ± 2.65

52

98

2

98

2.06

98 ± 3.61

51

98

2

96

2.08

115

-

51.3 ± 4.62

27

91

3

89

3.37

51.7 ± 5.69

27

92

2

90

2.22

120

-

18 ± 2.65

10

95

4

93

4.3

18 ± 1

10

98

2

95

2.11

Untreated control

 

+

201 ± 8.14

103

99

3

100

3

201.3 ± 4.16

101

95

3

96

3.13

Negative control

 

+

195.7 ± 5.51

100

100

4

101

3.96

200 ± 1

100

100

6

101

5.94

Positive control (DMBA)

20

+

130.3 ± 0.58

70

72

467

67

697.01

125 ± 5.57

68

69

462

67

689.55

Test item

20

+

185.3 ± 3.06

95

92

2

93

2.15

184 ± 3.61

92

96

5

97

5.15

60

+

168.7 ± 3.06

86

92

3

93

3.23

168 ± 6.24

84

95

2

96

2.08

100

+

148.3 ± 3.06

76

96

5

97

5.15

146.7 ± 3.51

73

97

6

98

6.12

120

+

126.7 ± 3.51

65

91

4

92

4.35

126.7 ± 3.79

63

96

3

97

3.09

140

+

88 ± 2.65

45

93

3

94

3.19

85.3 ± 3.51

43

94

2

95

2.11

160

+

45.3 ± 4.73

23

99

2

100

2

46 ± 4

23

96

2

97

2.06

170

+

24.7 ± 4.73

13

92

2

94

2.13

26.7 ± 4.51

13

96

4

96

4.17

Experiment 2 (20 h treatment)

 

 

culture I

 

culture II

Untreated control

 

-

184 ± 4.36

101

97

2

97

2.06

187 ± 5.29

101

99

2

99

2.02

Negative control

 

-

182.7 ± 4.04

100

100

2

100

2

185.7 ± 3.79

100

100

3

100

3

Positive control (EMS)

0.4

-

47.3 ± 6.11

26

58

398

58

686.21

50 ± 2.65

27

59

401

59

679.66

Test item

10

-

184.7 ± 2.31

101

96

2

96

2.08

180.7 ± 3.06

97

96

2

96

2.08

20

-

159.3 ± 1.15

87

94

2

94

2.13

161.3 ± 3.21

87

94

2

94

2.13

25

-

145.3 ± 2.08

80

95

3

95

3.16

150.3 ± 1.53

81

94

2

94

2.13

30

-

108 ± 2

59

94

2

94

2.13

102.3 ± 2.52

55

93

3

93

3.23

32.5

-

81.7 ± 6.43

45

102

4

102

3.92

83.3 ± 3.06

45

99

3

99

3.03

35

-

48.7 ± 3.51

27

91

3

91

3.3

44.7 ± 2.89

24

91

2

91

2.2

37.5

-

28.3 ± 4.16

15

95

2

91

2.2

33.3 ± 4.73

18

93

2

89

2.25

Experiment 2 (5 h treatment)

 

 

culture I

 

culture II

Untreated control

 

+

198 ± 6

101

102

2

97

2.06

200.3 ± 0.58

102

102

3

99

3.03

Negative control

 

+

195.3 ± 6.66

100

100

4

95

4.21

196 ± 2

100

100

4

97

4.12

Positive control (DMBA)

20

+

129 ± 11.53

71

72

468

72

650

125 ± 7.94

68

64

468

61

767.21

Test item

20

+

181 ± 6.56

93

105

3

100

3

185 ± 4.58

94

99

3

96

3.13

60

+

166.3 ± 4.04

85

102

3

97

3.09

165.3 ± 2.52

84

95

3

92

3.26

100

+

147.3 ± 1.53

75

99

4

94

4.26

141 ± 1

72

99

5

96

5.21

120

+

134 ± 3.61

69

100

4

95

4.21

133.3 ± 3.79

68

98

3

95

5.16

140

+

81.3 ± 5.13

42

103

4

98

4.08

82.7 ± 4.16

42

98

4

95

4.21

160

+

48.7 ± 7.51

25

103

2

98

2.04

45.7 ± 3.21

23

99

3

96

3.13

170

+

28.7 ± 5.03

15

95

3

90

3.33

32.3 ± 3.06

16

94

4

91

4.4

 

Conclusions:
The test item ditetradecyl peroxydicarbonate was not mutagenic in this in vitro mammalian cell gene mutation test performed with CHO-K1 (Chinese hamster ovary) cells.
Executive summary:

The test item, ditetradecyl peroxydicarbonate was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The test item was dissolved in N,N-dimethylformamide and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation using S9 mix).

Two independent main experiments (both run in duplicate) were performed at the concentrations and treatment intervals given below:

Experiment 1, 5-hour treatment period without S9 mix:

80, 90, 100, 105, 110, 115 and 120 µg/mL

Experiment 1, 5-hour treatment period with S9 mix:

20, 60, 100, 120, 140, 160 and 170 µg/mL

Experiment 2, 20-hour treatment period without S9 mix:

10, 20, 25, 30, 32.5, 35 and 37.5 µg/mL

Experiment 2, 5-hour treatment period with S9 mix:

20, 60, 100, 120, 140, 160 and 170 µg/mL

 

In Experiment 1, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no biologically differences between treatment and control groups and no dose-response relationships were noted.

 

In Experiment 2, the mutant frequency of the cells did not show significant alterations compared to the concurrent control, when the test item was tested without S9 mix over a prolonged treatment period (20 hours). Furthermore, a five-hour treatment with in the presence of S9 mix did not cause significant increases in mutant frequency, further indicating that the findings in Experiment 1 were within the normal biological variation.

 

As in Experiment 1, in Experiment 2 no statistical differences between treatment and solvent control groups and no dose response relationships were noted.

The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large increases in mutation frequency in the positive control cultures.

 

Ditetradecyl peroxydicarbonate tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this test in Chinese hamster ovary cells. Ditetradecyl peroxydicarbonate was not mutagenic in this in vitro mammalian cell gene mutation test performed with CHO-K1 cells.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-02-06 to 2012-04-20
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:
(1998, adopted July 21,1997)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
EC No.440/2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: In Vitro Mammalian Chromosome Aberration Test
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: DMEM:F12 (Dulbecco's modified eagle medium / Ham's F12 medium; mixture 1:1) already supplemented with 200 mM Glutamax. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 Ng/mL), the mitogen phytohemagglutinin (3 µg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES, and the anticoagulant heparin (25.000 IE/mL)
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-fraction of phenobarbital/ β-naphthoflavone induced male rats
Test concentrations with justification for top dose:
Range finder test:
Experiment I: Preparation interval 22 hrs with and without S9 mix: exposure period 4 hrs
8.7, 15.3, 26.7, 46.8, 81.8, 143.2, 250.6, 438.5, 767.3, 1342.9, 2350.0 µg/mL

Experiment II: Preparation interval 22 hrs without S9 mix: exposure period 22 hrs
8.7, 15.3, 26.7, 46.8, 81.8, 143.2, 250.6, 438.5, 767.3, 1342.9, 2350.0 µg/mL

Experiment II: Preparation interval 22 hrs with S9 mix: exposure period 4 hrs
26.7, 46.8, 81.8, 143.2, 250.6, 438.5, 767.3, 1342.9, 2350.0 µg/mL

Main test:
Based on the results of the range finder test the maximum concentration for each experiment group with and without S9 mix was the concentration were precipitation occurred at the end of treatment.

Experiment I: Preparation interval 22 hrs without S9 mix: exposure period 4 hrs
Positive control: EMS 770.0 µg/mL
Test item: 46.8, 81.8, 143.2 µg/mL

Experiment I: Preparation interval 22 hrs with S9 mix: exposure period 4 hrs
Positive control: CPA 7.5 µg/mL
Test item: 26.7, 46.8, 81.8 µg/mL

Experiment II: Preparation interval 22 hrs without S9 mix: exposure period 22 hrs
Positive control: EMS 550.0 µg/mL
Test item: 15.3, 26.7, 46.8 µg/mL

Experiment II: Preparation interval 22 hrs with S9 mix: exposure period 4 hrs
Positive control: CPA 2.5 µg/mL
Test item: 26.7, 46.8, 81.8 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: THF
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: In culture medium DMEM:F12

DURATION
- Exposure duration: Short term 4hrs with and without S9 mix; continuous 22 hrs without S9 mix
- Fixation time: 22 hrs

SPINDLE INHIBITOR: Colcemid
STAIN: Giemsa

NUMBER OF REPLICATION
Range finder test: 2 replicates/concentration
Main test: 2 replicates/concentration and experiment group

NUMBER OF CELLS EVALUATED:
Range finder test: 1000 cells per culture
Main test: 100 cells/concentration

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes
- Analysis of Metaphase Cells: Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. Only metaphases with 46 ± 1 centromer regions were included in the analysis.
Evaluation criteria:
Acceptability of the Assay

The chromosomal aberration assay is considered acceptable if it meets the following criteria:
a) The number of aberrations found in the solvent controls falls within the range of the historical aboratory control data .
b) The positive control substances should produce significant increases in the number of cells with structural chromosome aberrations.

Evaluation of Results

A test item is classified as non-mutagenic if:
-the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the historical control data .
-no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as mutagenic if:
-the number of induced structural chromosome aberrations is not in the range of the historical control data and
-either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the Fisher’s exact test. Evaluation was performed only for cells carrying aberrations excluding gaps.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS

- Effects of pH: No
- Effects of osmolality: No
- Evaporation from medium: No
- Water solubility: Insoluble
- Precipitation: Yes;
Without S9 mix: > 143.2 µg/mL
With S9 mix: >81.8 µg/mL
- Other confounding effects:No

RANGE-FINDING/SCREENING STUDIES

In in the range finder test precipitations were observed at the following concentrations.
Experiment I without S9 mix exposure period 4 hrs: 143.2 µg/mL
Experiment I with S9 mix exposure period 4 hrs: 81.8 µg/mL
Experiment II without S9 mix exposure period 22 hrs: 46.8 µg/mL
Experiment II with S9 mix exposure period 4 hrs: 81.8 µg/mL

In both experiments in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration of 2350.0 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA

Percentage of aberrant cells in human lymphocyte cultures (2009-2010)
Without S9 mix: Preparation interval 22 hrs, treatment 4 hrs
Organic Solvent: 0.0-3.0 %
EMS: 7.5-20.0 %

Without S9 mix: Preparation interval 22 hrs, treatment 22 hrs
Organic Solvent: 0.0-3.0%
EMS: 7.5 – 53.0 %

With S9 mix: Preparation interval 22 hrs, treatment 4 hrs
Organic Solvent: 0.0-3.0 %
CPA: 7.5 – 40.0 %

The aberration rates of the cells after treatment with the test item (0.5 - 2.5 % aberrant cells, excluding gaps) were close to the range of the solvent control values (0.5 - 2.0% aberrant cells, excluding gaps) and within the range of the laboratory historical solvent control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY

The mitotic index in two cultures (1000 cells per culture) in each concentration with and without S9 was determined.
Using reduced mitotic indices as an indicator for toxicity in Experiment I, no cytotoxic effects were observed after 4 hours treatment in the absence and presence of S9 mix. Therefore, 2350.0 µg/mL was chosen as top treatment concentration for Experiment II.

OTHER OBSERVATION:
Polyploidy : No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

 

Experiment

Preparation interval [h]

Test item concentration [µg/mL]

Mitotic indices in %

Aberrant cells in %

 of control

incl. gaps

excl. gaps

carrying exchanges

Exposure period 4 h without S9 mix

1

22

Solvent control (THF 0.5 % (v/v))

100

0.5

0.5

0

Positive control (EMS 770.0)

66.7

9

8.5 S

3

46.8

112.6

0.5

0.5

0.5

81.8

120.7

0.5

0.5

0

143.2 P

100.4

0.5

0.5

0

Exposure period 22 h without S9 mix

2

22

Solvent control (THF 0.5 % (v/v))

100

1

1

0

Positive control (EMS 550.0)

35.7

11

11 S

2

15.3

84.5

1

1

0

26.7

92.4

2

2

0

46.8 P

84.9

2

2

0

Exposure period 4 h with S9 mix

1

22

Solvent control (THF 0.5 % (v/v))

100

0.5

0.5

0

Positive control (CPA 7.5)

77.2

7.5

7.5 S

1

26.7

78.8

0.5

0.5

0

46.8

90

2.5

2.5

0

81.8 P

87.6

1

1

0

2

22

Solvent control (THF 0.5 % (v/v))

100

2

2

0

Positive control (CPA 2.5)

92

12.5

12.5 S

1

26.7

101.3

1

1

0

46.8

102

3

2

0

81.8 P

97.7

1

1

0

* = Including cells carrying exchanges

P = Precipitation occurred at the end of treatment

S = Aberration frequency statistically significant higher than corresponding control values

Conclusions:
The test item Dimyristylperoxydicarbonate is considered to be non-clastogenic in this chromosome aberration test, when tested up to precipitating concentrations.
Executive summary:

The test item Dimyristylperoxydicarbonate, dissolved in THF, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix. The test was conducted according to the OECD guideline No.473 (1998) and Commission Regulation (EC) No.440/2008 B.10(2008).

Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without S9 mix. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours (Exp. I & II) after the start of treatment with the test item. In each experimental group two parallel cultures were analysed. 100 metaphases per culture were scored for structural chromosomal aberrations. 1000 cells were counted per culture for determination of the mitotic index. The highest treatment concentration in this study, 2350.0 µg/mL was chosen with regard to the solubility properties of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests. In Experiment I, visible precipitation of the test item in the culture medium was observed at 143.2 µg/mL and above in the absence of S9 mix and at 81.8 µg/mL and above in the presence of S9 mix. In addition, precipitation occurred in Experiment II, in the absence of S9 mix, at 46.8 µg/mL and above and in the presence of S9 mix at 81.8 µg/mL and above at the end of treatment.

No relevant influence on osmolarity or pH value was observed. In this study, in the absence as well as in the presence of S9 mix, no biologically relevant cytotoxicity indicated by clearly reduced mitotic indices could be observed. In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.5 - 2.5 % aberrant cells, excluding gaps) were close to the range of the solvent control values (0.5 - 2.0% aberrant cells, excluding gaps) and within the range of the laboratory historical solvent control data. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. In both experiments, either EMS (550 or 770 µg/mL) or CPA (2.5 or 7.5 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item Dimyristylperoxydicarbonate did not induce structural chromosomal aberrations in human lymphocytes in vitro, when tested up to precipitating concentrations.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames Test :

The test item Dimyristylperoxydicarbonate was tested for mutagenic activity with the "Salmonella typhimurium Reverse Mutation Test" (Ames Test). The study was conducted in accordance with the OECD-guideline 471 and the Council Regulation (EC) No 440/2008, Method B.13/14.

The test substance was dissolved in acetone. The following concentrations were tested: In the first experiment: 21, 62, 185, 556 and 1667 μg per plate without external metabolisation, and 21, 62, 185, 556 and 1667 μg per plate with S9-mix from Aroclor 1254 induced microsomes of rat liver as an external metabolising system.

In the second experiment: 7, 21, 62, 185 and 556 μg per plate without external metabolisation, and 7, 21, 62, 185 and 556 μg per plate with S9-mix from Aroclor 1254 induced microsomes of rat liver as an external metabolising system. In the first experiment the test was performed according to the "direct plate incorporation method", in the second experiment according to the "preincubation method".

As test system the bacterial strains Salmonella typhimurium TA 97a, TA 98, TA 100, TA 102 and TA 1535 were used. Negative and positive controls were included. According to the results obtained in this study, Dimyristylperoxydicarbonate is nonmutagenic in the Ames test with the strains TA 97a, TA 98, TA 100, TA 102 and TA 1535 with and without an external metabolising system up to 1667 μg/plate, which is the limit of solubility.

 

HPRT Test:

The test item Dimyristylperoxydicarbonate was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The test item was dissolved in N,N-dimethylformamide and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation using S9 mix).

 

Two independent main experiments (both run in duplicate) were performed at the concentrations and treatment intervals given below:

Experiment 1, 5-hour treatment period without S9 mix:

80, 90, 100, 105, 110, 115 and 120 µg/mL

Experiment 1, 5-hour treatment period with S9 mix:

20, 60, 100, 120, 140, 160 and 170 µg/mL

Experiment 2, 20-hour treatment period without S9 mix:

10, 20, 25, 30, 32.5, 35 and 37.5 µg/mL

Experiment 2, 5-hour treatment period with S9 mix:

20, 60, 100, 120, 140, 160 and 170 µg/mL

 

In Experiment 1, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no biologically differences between treatment and control groups and no dose-response relationships were noted.

In Experiment 2, the mutant frequency of the cells did not show significant alterations compared to the concurrent control, when the test item was tested without S9 mix over a prolonged treatment period (20 hours). Furthermore, a five-hour treatment with in the presence of S9 mix did not cause significant increases in mutant frequency, further indicating that the findings in Experiment 1 were within the normal biological variation.

As in Experiment 1, in Experiment 2 no statistical differences between treatment and solvent control groups and no dose response relationships were noted.

The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large increases in mutation frequency in the positive control cultures.

The test item tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this test in Chinese hamster ovary cells. The test item was not mutagenic in this in vitro mammalian cell gene mutation test performed with CHO-K1 cells.

 

Chromosome Aberration test

The test item Dimyristylperoxydicarbonate, dissolved in THF, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix. The test was conducted according to the OECD guideline No.473 (1998) and Commission Regulation (EC) No 440/2008 B.10(2008).

Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without S9 mix. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours (Exp. I & II) after the start of treatment with the test item. In each experimental group two parallel cultures were analysed. 100 metaphases per culture were scored for structural chromosomal aberrations. 1000 cells were counted per culture for determination of the mitotic index. The highest treatment concentration in this study, 2350.0 µg/mL was chosen with regard to the solubility properties of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests. In Experiment I, visible precipitation of the test item in the culture medium was observed at 143.2 µg/mL and above in the absence of S9 mix and at 81.8 µg/mL and above in the presence of S9 mix. In addition, precipitation occurred in Experiment II, in the absence of S9 mix, at 46.8 µg/mL and above and in the presence of S9 mix at 81.8 µg/mL and above at the end of treatment.

No relevant influence on osmolarity or pH value was observed. In this study, in the absence as well as in the presence of S9 mix, no biologically relevant cytotoxicity indicated by clearly reduced mitotic indices could be observed. In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.5 - 2.5 % aberrant cells, excluding gaps) were close to the range of the solvent control values (0.5 - 2.0% aberrant cells, excluding gaps) and within the range of the laboratory historical solvent control data. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. In both experiments, either EMS (550 or 770 µg/mL) or CPA (2.5 or 7.5 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item Dimyristylperoxydicarbonate did not induce structural chromosomal aberrations in human lymphocytes in vitro, when tested up to precipitating concentrations.

Justification for classification or non-classification

Based on the experimental results the test substance is not classified and labelled with respect to genetic toxicity according to Regulation (EC) No 1272/2008 (CLP).