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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Tert-Butylperoxy-3,5,5-trimethylhexanoat was tested for genetic toxicity in four in vitro tests. The test substance showed mutagenic activity in bacterial cells (Ames test according to OECD guideline 471 and EU method B.13/14, with metabolic activation (S9 mix)) but not in V79 cells (HPRT test according to OECD guideline 476 and EU method B.17, with and withoud metabolic activation (S9 mix)). Furthermore, the test substance induced chromosome aberrations in cultured human lymphocytes with metabolic activation (S9 mix) in two in vitro mammalian chromosome aberration tests according to OECD guideline 473 and EU method B.10.

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:
04 June 2020 - 28 July 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997, revised June 2020
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
The Salmonella typhimurium and E.coli histidine (his) reversion system measures his- -> his+ reversions. The strains are constructed to differentiate between base pair (E.coli WP2, TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital- and ß-naphthoflavone-induced rat liver S9 mix
Test concentrations with justification for top dose:
Concentrations:
Preliminary concentration range finding test: TA98 and TA100,+S9 and -S9: 5, 16, 50, 160, 500, 1600, 5000 µg/plate;

Main experiment I (initial mutation test): all strains:
-S9: 5, 16, 50, 160, 500, 1600, 5000 µg/plate; +S9: 16, 50, 100, 160, 250, 500, 1000 µg/plate;

Main experiment II (confirmatory mutation test): all strains (+S9): 50, 80, 100, 160, 250, 320, 400 µg/plate;

Justification for top dose: tested up to recommended maximum test concentration according to OECD 471 in the preliminary concentration range finding tes; due to observed toxicity lower concentrations were chosen for main experiments 1 and 2
Vehicle / solvent:
- Vehicle/solvent used: DMSO (test item, positive controls), Ultrapure water (positive controls)
- Justification for choice of solvent/vehicle: In a non-GLP pre-test, the solubility of the test item was determined. Based on these results, DMSO was chosen as solvent for the test item.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
50 µg/plate (E.coli WP2, +S9) 2 µg/plate (all Salmonella strains, +S9)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ultrapure water
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
2 µL/plate (E.coli WP2, -S9)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
50 µg/plate (TA1537, -S9)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ultrapure water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
2 µg/plate (TA100, TA1535, -S9)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-1,2-phenylenediamine
Remarks:
4 µg/plate (TA98, -S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
plate incorporation

TREATMENT:
preliminary concentration range finding test and main experiment 1 both with and without S9 mix and main experiment 2 with S9 mix):
- test substance solution (0.1 mL)
- S9 mix (0.5 mL) or phosphate buffer (0.5 mL)
- bacterial suspension (0.1 mL)
were mixed with 2 mL of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: Determination of revertant colony number and the background lawn of auxotrophic cells of two test strains
Evaluation criteria:
The mean values and standard deviations of each threefold determination are calculated as well as the increase factor of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (mean revertants minus mean spontaneous revertants) is given. A substance is considered to have mutagenic potential, if a reproducible increase of re-vertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed . A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.
Statistics:
Not performed as not mandatory for this test system.
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: no precipitation observed at any concentratration level

RANGE-FINDING/SCREENING STUDIES:
A strong inhibitory, cytotoxic effect of the test item was observed in both investigated strains (TA98+TA100) down to and including the concentration level of 500 μg/plate, in the presence of exogenous metabolic activation (+S9).

STUDY RESULTS
- Concurrent vehicle negative and positive control data
See tables in "Any other information on results incl. tables"

Ames test:
- Signs of toxicity
- Individual plate counts
- Mean number of revertant colonies per plate and standard deviation
See tables in "Any other information on results incl. tables"

Table 1: Summary Table of the Results of the Concentration Range Finding Test

 

Strain

TA 98

TA 98

TA 98

TA 98

TA 100

TA 100

TA 100

TA 100

Induction

-S9

-S9

+S9

+S9

-S9

-S9

+S9

+S9

Mean values of revertants per plate and
Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

22.7

1.03

25.0

1.12

80.3

1.05

105.7

1.15

DMSO Control

22.0

1.00

22.3

1.00

76.7

1.00

91.7

1.00

Ultrapure Water Control

79.7

1.00

5000 µg/plate

17.0

0.77

0.0

0.00

67.3

0.88

0.0

0.00

1600 µg/plate

14.0

0.64

0.0

0.00

92.0

1.20

0.0

0.00

500 µg/plate

21.3

0.97

8.7

0.39

67.0

0.87

81.3

0.89

160 µg/plate

20.7

0.94

72.3

3.24

69.3

0.90

202.3

2.21

50 µg/plate

14.7

0.67

27.3

1.22

73.0

0.95

94.7

1.03

16 µg/plate

19.0

0.86

23.3

1.04

70.0

0.91

81.0

0.88

5 µg/plate

20.0

0.91

19.7

0.88

59.3

0.77

86.0

0.94

NPD (4 µg/plate)

342.0

15.55

SAZ (2 µg/plate)

1045.3

13.12

2AA (2 µg/plate)

1724.0

77.19

1730.7

18.88

MR: Mutation Rate

NPD: 4-Nitro-1,2-phenylenediamine

SAZ: Sodium azide

2AA: 2-aminoanthracene

Remarks: DMSO was applied as vehicle of the test item and the positive control substances NPD and 2AA. The ultrapure water was applied as vehicle of the positive control substance SAZ. The mutation rate obtained at the test item, at the untreated control; furthermore, at NPD and 2AA refers to the DMSO. The mutation rate obtained at SAZ refers to ultrapure water.;

 

Table 2: Summary Table of the Results of the Main experiment 1(Initial Mutation Test)

Strain

TA 98

 

 

 

TA 100

 

 

 

TA 1535

 

 

 

TA 1537

 

 

 

WP2 uvrA

 

 

 

Induction

-S9

-S9

+S9

+S9

-S9

-S9

+S9

+S9

-S9

-S9

+S9

+S9

-S9

-S9

+S9

+S9

-S9

-S9

+S9

+S9

Mean values of revertants per plate Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

21.7

0.93

24.0

1.01

74.7

0.93

92.7

0.95

11.7

1.03

13.3

1.11

6.0

1.13

8.3

1.14

50.7

1.06

54.0

1.08

DMSO Control

23.3

1.00

23.7

1.00

80.0

1.00

98.0

1.00

11.3

1.00

12.0

1.00

5.3

1.00

7.3

1.00

47.7

1.00

50.0

1.00

Ultrapure Water Control

76.7

1.00

8.3

1.00

55.0

1.00

5000 µg/plate

24.0

1.03

71.7

0.90

14.0

1.24

6.3

1.19

52.0

1.09

1600 µg/plate

25.0

1.07

98.3

1.23

13.7

1.21

7.7

1.44

51.3

1.08

1000 µg/plate

0.0

0.00

0.0

0.00

0.0

0.00

0.0

0.00

0.0

0.00

500 µg/plate

24.3

1.04

0.0

0.00

76.0

0.95

9.0

0.09

10.0

0.88

0.0

0.00

7.0

1.31

10.3

1.41

49.0

1.03

85.3

1.71

250 µg/plate

62.7

2.65

117.3

1.20

12.0

1.00

30.7

4.18

183.7

3.67

160 µg/plate

19.3

0.83

87.3

3.69

68.0

0.85

199.0

2.03

8.7

0.76

13.0

1.08

4.3

0.81

24.3

3.32

44.7

0.94

185.3

3.71

100 µg/plate

78.3

3.31

176.7

1.80

9.3

0.78

26.0

3.55

160.3

3.21

50 µg/plate

19.0

0.81

30.3

1.28

71.3

0.89

105.3

1.07

9.7

0.85

11.7

0.97

7.0

1.31

9.3

1.27

37.0

0.78

94.7

1.89

16 µg/plate

16.7

0.71

23.0

0.97

67.7

0.85

76.7

0.78

8.7

0.76

10.7

0.89

4.3

0.81

9.0

1.23

34.7

0.73

50.0

1.00

5 µg/plate

15.0

0.64

71.7

0.90

10.0

0.88

5.0

0.94

31.0

0.65

NPD (4 µg/plate)

418.7

17.94

SAZ (2 µg/plate)

1000.0

13.04

1314.7

157.76

9AA (50 µg/plate)

793.3

148.75

MMS (2 mL/plate)

1131.7

20.58

2AA (2 µg/plate)

1184.0

50.03

1341.3

13.69

112.3

9.36

85.3

11.64

2AA (50 µg/plate)

171.3

3.43

MR: Mutation Rate;     NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene

Remarks:       DMSO was applied as vehicle of the test item and the positive control substances NPD, 9AA and 2AA. The ultrapure water was applied as vehicle of the positive control substances SAZ and MMS. The mutation rate obtained at the test item, at the untreated control; furthermore, at NPD, 9AA and 2AA refers to the DMSO. The mutation rate obtained at SAZ and MMS refers to ultrapure water.

 

Table 3: Summary Table of the Results of the main experiment 2 (Confirmatory Mutation Test)

Strain

TA 98

 

 

 

TA 100

 

 

 

TA 1537

 

 

 

WP2 uvrA

 

 

 

Induction

-S9

-S9

+S9

+S9

-S9

-S9

+S9

+S9

-S9

-S9

+S9

+S9

-S9

-S9

+S9

+S9

Mean values of revertants per plate Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

28.7

1.12

86.7

0.90

7.7

1.28

49.7

1.39

DMSO Control

25.7

1.00

96.3

1.00

6.0

1.00

35.7

1.00

400 µg/plate

18.3

0.71

61.0

0.63

17.7

2.94

109.0

3.06

320 µg/plate

29.7

1.16

106.0

1.10

21.7

3.61

154.3

4.33

250 µg/plate

61.7

2.40

156.0

1.62

27.0

4.50

177.3

4.97

160 µg/plate

76.0

2.96

200.0

2.08

26.3

4.39

180.7

5.07

100 µg/plate

63.3

2.47

173.7

1.80

17.3

2.89

147.0

4.12

80 µg/plate

49.7

1.94

139.3

1.45

10.7

1.78

109.3

3.07

50 µg/plate

27.3

1.06

101.0

1.05

6.7

1.11

76.7

2.15

2AA (2 µg/plate)

1234.7

48.10

1386.7

14.39

100.3

16.72

2AA (50 µg/plate)

156.3

4.38

MR: Mutation Rate;  2AA: 2-aminoanthracene

Remarks:   DMSO was applied as vehicle of the test item and the positive control substance 2AA. The mutation rate obtained at the test item, at the untreated control; furthermore, at 2AA refers to the DMSO.

Conclusions:
Under the experimental conditions, the test substance (acting as promutagen, in presence of exogenous metabolic activation) induced gene mutations by frameshift and base-pair substitution in the genome of Salmonella typhimurium TA98, TA100, TA1537 and Escherichia coli WP2 strains. Therefore, TBPIN is considered mutagenic in this bacterial reverse mutation assay.
Executive summary:

To investigate the mutagenic potential of TBPIN, five bacterial strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA were used in a plate incorporation test (main experiment I, initial mutation test) and this test item was further investigated with Salmonella typhimurium TA98, TA100, TA1537 and Escherichia coli WP2 uvrA strains in a repeated plate incorporation test (experiment II, confirmatory mutation test). The initial mutation test was conducted with and without metabolic activation (±S9), the confirmatory mutation test was performed with addition of metabolic activation (+S9), only. The concentrations, including the controls, were tested in triplicate (positive and negative controls were run concurrently). In the performed experiments all of the validity criteria, regarding the investigated strains, negative (vehicle) and positive controls, S9 activity and number of investigated analysable concentration levels were fulfilled. In the initial mutation test following treatment with TBPIN significant, biological relevant revertant colony number increases, positive results were obtained (in Salmonella typhimurium TA98, TA100, TA1537 and Escherichia coli WP2 uvrA strains) in the presence of exogenous metabolic activation (+S9). The dose related tendencies, the unequivocal positive results were repeated, adequately confirmed in a subsequent experiment: in the confirmatory mutation test (repeated plate incorporation test) in TA100, TA1537 and in Escherichia coli WP2 uvrA, and borderline results were obtained in TA98. In the performed experiments inhibitory effect of the test item (indicated by affected revertant colony numbers (revertants below the vehicle control data range and/or below the corresponding historical control data range) and/or affected background lawn development: absent, reduced or slightly reduced background lawn development) was observed in all examined strains. The 250 μg/plate was found to be the lowest cytotoxic concentration, observed in the initial mutation test, in the case of Salmonella typhimurium TA100 strain, in the presence of exogenous metabolic activation (+S9). No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9).

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-01-04 to 2010-02-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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:
other: mammalian cell gene mutation assay
Target gene:
The V79 cells are exposed to the test item both with and without metabolic activation. The study was determined whether the test item or its metabolites can induce forward mutation at the hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt) in cultured Chinese hamster cells.
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Test concentrations with justification for top dose:
Two independent main experiments (both run in duplicate) were performed at the concentrations and treatment intervals given below:
Experiment 1 (3-hour treatment period without and with S9 mix): 9.76, 19.53, 39.06, 78.12, 156.25, 234.37 and 312.50 µg/mL
Experiment 2 (20-hour treatment period without S9 mix): 9.76, 19.53, 39.06, 78.12, 156.25, 234.37 and 312.50 µg/mL
Experiment 2 (3-hour treatment period with S9 mix): 9.76, 19.53, 39.06, 78.12, 156.25, 234.37 and 312.50 µg/mL
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
The test item, tert. butylperoxy-3,5,5-trimethylhexanoate (TBPIN) was tested in a Mammalian Gene Mutation Test in V79 cells. The test item was dissolved in DMSO and the used concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation). Two independent main experiments (both run in duplicate) were performed.
Evaluation criteria:
The mutation frequency was calculated by dividing the total number of mutant colonies by the number of cells selected (10e6 cells: 5 plates at 2 x 10e5 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection (viability), and was expressed as 6-TG resistant mutants per 10e6 cloneable cells. Assay acceptance criteria:
- The mutant frequency in the negative (solvent) control cultures is in line with the historical control data.
- The positive control chemicals induce a clear increase in mutant frequency.
- The cloning efficiency of the negative controls is between the range of 60% to 140% on Day 0 and 70% to 130% on Day 7.
Statistics:
Wilcoxon-Mann-Whitney U-test was used to determine whether or not there are statistically significant increases in mutant frequency.
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:
not applicable
Positive controls validity:
valid
Additional information on results:
In Experiment 1, there were no toxicologically significant increases in mutant frequency at any concentration, either in the absence or in the presence of metabolic activation. There were no statistical 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 examined without S9 mix over a prolonged treatment period (20 hours). Further, a 3-hour treatment in the presence of S9 mix did not cause increases in mutant frequency, further confirming the findings in Experiment 1.
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.
Conclusions:
The test item tert-Butylperoxy-3,5,5-trimethylhexanoat was not mutagenic in this in vitro mammalian cell gene mutation test performed with V79 (Chinese hamster lung) cells, even tested at clearly cytotoxic concentrations.
Executive summary:

The test item tert-Butylperoxy-3,5,5-trimethylhexanoat was tested in a Mammalian Gene Mutation Test in V79 cells per EU-method B.17. The test item was dissolved in DMSO and this concentrations were selected based on the results of the preliminary study (without and with metabolic activation): 9.76, 19.53, 39.06, 78.12, 156.25, 234.37 and 312.50 ug/mL. Two independent main experiments were performed. In Experiment 1, there were no biologically or statistically significant increases in mutation frequency at any concentration, either in the absence or in the presence of metabolic activation. In Experiment 2, the mutant frequency of the cells did not show significant alterations compared to the concurrent control, when examined without S9 mix over a prolonged treatment period of 20 hours. In addition, a 3-hour treatment in the presence of S9 mix did not cause 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 control groups and no dose-response relationships were noted. Tert-Butylperoxy-3,5,5-trimethylhexanoat tested both without and with metabolic activation (S9 mix), did not include increases in mutant frequency in this test in Chinese hamster lung cells. Therefore, tert-Butylperoxy-3,5,5-trimethylhexanoat was not mutagenic (tox. (survival) ~100 % - 15%) in this in vitro mammalian cell gene mutation test performed with V79 cells.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002-03-06 to 2002-07-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Type of assay:
other: in vitro mammalian chromosome aberration assay
Species / strain / cell type:
lymphocytes: primary culture
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
The S9 mix consists of induced enzymatic systems contained in rat liver post-mitochondrial fraction (S9 fraction) and the cofactors necessary for their function.
Test concentrations with justification for top dose:
The test item was dissolved in the vehicle at concentrations of:
- 460.66 and 23.03 mg/mL for the first experiment,
- 38.234 and 17.274 mg/mL for the second experiment,
- 38.234 mg/mL for the third experiment.
The preparations were made immediately before use.
Vehicle / solvent:
- Vehicle/solvent used: dimethylsulfoxide (DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
was used at the experiments without S9 mix and at a final concentration of 3 µg/mL, 3 hours of treatment) or 0.2 µg/mL (continuous treatment)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
was used at the experiments with S9 mix and at a final concentration of 50 µg/mL and 25 µg/mL. Tthe dose-level which gave a satisfactory response in terms of quality and quantity of methaphases and extent of chromosomal damage was selected.
Details on test system and experimental conditions:
In the first experiment, lymphocyte cultures were exposed to the test or control items, with or without S9 mix, for 3 hours then rinsed. Cells were harvested 20 hours after 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 until harvest to the test or control items,
- with S9 mix, cells were exposed to the test or control items for 3 hours and then rinsed.
Cells were harvested 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later, respectively.
The third experiment with S9 mix was performed under the same experimental conditions of the first experiment.
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.
Key result
Species / strain:
lymphocytes: primary culture
Metabolic activation:
with and without
Genotoxicity:
other: genotoxic only with metabolic activation (S9 mix)
Cytotoxicity / choice of top concentrations:
other: ambiguous
Vehicle controls validity:
not examined
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
In the culture medium, the dose-level of 10 mM (corresponding to 2303.3 µg/mL) showed a moderate emulsion. At this dose-level, the pH and the osmolality values were equivalent to those of the vehicle control culture.
With a treatment volume of 27.5 µL/ 5.5 mL culture medium, the treatment-levels were as follows:
- 0.16, 0.31, 0.63, 1.25, 2.5, 5, 7.5 and 10 mM, for the first experiment with and without S9 mix,
In the experiment without S9 mix the dose-levels being too toxic, the treatment was repeated at the following dose-levels:
- 0.031, 0.063, 0.13, 0.25, 0.375 and 0.5 mM, for the first experiment without S9 mix (retained experiment),
- 0.016, 0.031, 0.063, 0.13, 0.25 and 0.375 mM, for the second experiment without S9 mix,
- 0.08, 0.16, 0.31, 0.47, 0.63 and 0.83 mM, for the second experiment with S9 mix,
- 0.20, 0.26, 0.35, 0.47, 0.62 and 0.83 mM, for the third experiment with S9 mix.
A slight to marked emulsion was observed at the end of the treatment period, at dose-levels >= 1.25 mM.

Experiment without S9 mix:
Cytotoxicity:
A slight to moderate toxicity was induced at dose-levels >= 0.13 mM, depending on the treatment duration.
Metaphase analysis:
The dose-levels selected for metaphase analysis were as follows:
- 0.13, 0.25 and 0.375 mM, for the 3 and 20-hour treatments,
- 0.375 mM, for the 44-hour treatment.

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 strong toxicity was generally noted at dose-levels >= 0.47 mM.

Metaphase analysis:
The dose-levels selected for metaphase analysis were as follows:
- 0.16, 0.31 and 0.63 mM, for the 20-hour harvest time in the first experiment,
- 0.47, 0.63 and 0.83 mM, for the 20-hour harvest time in the second and third experiments,
0.63 mM, for the 44-hour harvest time.

A significant increase in the frequency of cells with structural chromosome aberrations was noted at dose-levels >= 0.31 mM, at the 20-hour harvest time.
No significant increase in the frequency of cells with structural chromosomal aberrations was noted at the 44-hour harvest time.
The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were specified in acceptance criteria. The study was therefore considered valid.
Conclusions:
Under this experimental conditions, the test item tert-Butylperoxy-3,5,5-trimethylhexanoat induced chromosome aberrations in cultured human lymphocytes, with metabolic activation (S9 mix).
Executive summary:

Tert-Butylperoxy-3,5,5-trimethylhexanoat was tested in cultured human lymphocytes according to OECD guideline 473 (1997) and EU method B.10 (2000). The test item was tested in two independent experiments, both with and without a liver metabolising system (S9 mix), obtained from rats previously treated with Aroclor 1254.

With a treatment volume of 27.5 µL/ 5.5 mL culture medium, the treatment-levels were as follows:

- 0.16, 0.31, 0.63, 1.25, 2.5, 5, 7.5 and 10 mM, for the first experiment with and without S9 mix,

In the experiment without S9 mix the dose-levels being too toxic, the treatment was repeated at the following dose-levels:

- 0.031, 0.063, 0.13, 0.25, 0.375 and 0.5 mM, for the first experiment without S9 mix (retained experiment),

- 0.016, 0.031, 0.063, 0.13, 0.25 and 0.375 mM, for the second experiment without S9 mix,

- 0.08, 0.16, 0.31, 0.47, 0.63 and 0.83 mM, for the second experiment with S9 mix,

- 0.20, 0.26, 0.35, 0.47, 0.62 and 0.83 mM, for the third experiment with S9 mix.

A slight to marked emulsion was observed at the end of the treatment period, at dose-levels >= 1.25 mM.

Experiment without S9 mix:

Cytotoxicity:

A slight to moderate toxicity was induced at dose-levels >= 0.13 mM, depending on the treatment duration.

Metaphase analysis:

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 strong toxicity was generally noted at dose-levels >= 0.47 mM.

Metaphase analysis:

A significant increase in the frequency of cells with structural chromosome aberrations was noted at dose-levels >= 0.31 mM, at the 20-hour harvest time.

No significant increase in the frequency of cells with structural chromosomal aberrations was noted at the 44-hour harvest time.

The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were specified in acceptance criteria. The study was therefore considered valid.

Under this experimental conditions, the test item tert-Butylperoxy-3,5,5-trimethylhexanoat induced chromosome aberrations in cultured human lymphocytes, with metabolic activation (S9 mix).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

To clarify the clastogenic potential, an in vivo mammalian erythrocyte micronucleus test (MN test) according to OECD guideline 474 and EU method B12 was performed. Under the conditions of this assay, the test item did not induce any statistically and biologically relevant increase in the number of micronucleated poychromatic erythrocytes and is therefore considered not to be clastogenic in vivo.


To clarify the positive result of the Ames Test, a Comet assay in vivo was proposed.


 

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-04-12 to 2012-08-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: micronucleus assay in vivo
Species:
mouse
Strain:
other: CRL: NMRI BR
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: TOXI COOP Zrt. 1103 Budapest, Cserkesz u. 90.
- Age at study initiation: Young adult mice (7 -8 weeks)
- Weight at study initiation: 28.0g - 32.4 g
- Assigned to test groups randomly: Yes
- Fasting period before study: no
- Housing: Group caging (up to 2 and 5 animals/cage)
- Diet: ssniff® SM R/M-Z+H complete diet for rats and mice, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 %
- Air changes (per hr): 12 air exchanges per hour by central air-condition system
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 2012-04-11 To: 2012-04-13
Route of administration:
oral: gavage
Vehicle:
- Vehicle/solvent used: sunflower oil
- Amount of vehicle (if gavage or dermal): 10 mL/kg
- positive control: Cyclophosphamide (positive control) was dissolved in aqua ad iniectabilia for treatment
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The necessary amount of the test item was weighed into a calibrated volumetric flask and vehicle added and stirred to obtain homogenous formulations. It was then diluted to the final volume with vehicle. The concentration of the test item solutions were chosen to assure the same dosing volumes in mice for all dose levels (10 mL/kg).

CRITERIA FOR DOSE SELECTION:
A preliminary toxicity test was performed to identify the appropriate maximum dose level for the main test. The preliminary toxicity test determined whether there are differences in toxicity between the sexes or not. Groups of two male and female mice were treated on one occasion by oral gavage at dose levels of 2000 mg/kg bw. The treatment volume was 10 mL/kg bw. Animals were examined regularly for toxic signs and mortalities. On the basis of results of preliminary toxicity test, doses for the Mouse Micronucleus Test were: 500, 1000 and 2000 mg/kg bw.
Duration of treatment / exposure:
single dosage
Frequency of treatment:
single dosage
Post exposure period:
- untreated control, low and mid dose groups the sampling from bone marrow was performed once at 24 hours after treatment;
- high dose and the negative control groups the sampling from bone marrow was performed twice at 24 and 48 hours after treatment
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
untreated control, low and mid dose group, positive control group: 5 male rats
negative control group and hogh dose group: 10 male rats (5 rate for the 24h sampling time, 5 rats for the 48h sampling time)
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Positive control(s):
- Cyclophosphamide
- Route of administration: intraperitoneally
- Doses / concentrations: 10 mL/kg bw (60 mg/kg bw)
Tissues and cell types examined:
Please refer to details of tissue and slide preparation
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES:
The animals from each group were weighed and bone marrow was obtained from two exposed femurs of the mice from every dose- time point immediately after sacrificing. The bone marrow was flushed with foetal bovine serum (5 mL). After vortex mixing, the cell suspension was concentrated by centrifugation and the supernatant was discarded. Smears of the cell pellet were made on standard microscope slides. Slides were then dried at room temperature.

DETAILS OF SLIDE PREPARATION:
Subsequently the slides were stained as follow:
Fixed for a minimum of 5 minutes in methanol and allowed to air-dry.
1. Stained with Giemsa solution for 25 minutes.
2. Rinsing in distilled water.
3. Drying at room temperature (at least 12 hours).
4. Coating with EZ-mounting

METHOD OF ANALYSIS:
Prior to microscopic analysis, one slide from each animal was given a code number for blind microscopic analysis. The code labels were covering the original animal numbers to ensure that the slides scored without bias. Two thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells. The frequency of micronucleated cells were expressed as percent of micronucleated cells based on the first 2000 PCEs counted in the optic field.
The proportion of immature among total (immature + mature) erythrocytes was determined for each animal by counting a total of at least 200 immature erythrocytes.
Evaluation criteria:
A micronucleus is defined in following way:
– A bluish mauve strongly coloured uniform circular particle in the cell.
– The particle should have a certain size and it should be located inside the cells.
– During focusing, the particle should stay uniform in colour /light refraction and shape within a large interval.
– Cells with two or more micronuclei were counted as single micronucleated cells.

The Micronucleus Test is considered acceptable if it meets the following criteria:
– The proportion of polychromatic erythrocytes among total erythrocytes in treated groups is not less than 20 % of the control value.
– the frequencies of micronucleated polychromatic erythrocytes found in the negative and /or solvent controls are consistent with the range of historical laboratory control data.
– the positive control item should produce biologically relevant increases in the number of micronucleated polychromatic erythrocytes.
– Each treated and control group should include at least 5 analysable animals

The test item would have been considered to have shown genotoxic activity in this study if the following criteria are met:
– increases in the frequency of micronucleated polychromatic erythrocytes are observed in treated animals of a single dose group compared to the corresponding negative controls
– the increases are dose-related
– the increases are statistically significant.
– the increases exceeded historical control range for this laboratory

The test item was considered to have given a negative response because no reproducible, statistically significant increases were observed
Statistics:
The frequencies of micronucleated polychromatic erythrocytes in animals in the test and positive control groups were compared to the values found in the corresponding negative control group. Statistically analysis was performed using Kruskal Wallis Non Parametric ANOVA test (Kruskal-Wallis one-way analysis of variance).
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
clinical signs at the highest dose tested.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range:
Groups of two male and female mice were treated on one occasion by oral at dose levels of 2000 mg/kg bw.
- Clinical signs of toxicity in test animals:
The male mice dosed at 2000 mg/kg body weight showed medium, slight to medium decrease in activity and piloerection between 2 and 5 hours after the treatment. The female mice dosed at 2000 mg/kg body weight showed slight decrease in activity and piloerection between 2 and 4 hours after the treatment.
- Other:
No adverse reactions to treatment were observed in the male and female mice dosed 2000 mg/kg body weight between 5 and 48 hours after the treatment.

RESULTS OF DEFINITIVE STUDY

- Induction of micronuclei:
The frequencies of MPCEs for the untreated, negative (solvent) controls mice were within an acceptable range and compatible with the historical control data for this laboratory. Cyclophosphamide treated mice showed a large, statistically significant increase in the MPCEs number compared to the negative control, demonstrating an acceptable sensitivity of the test. The mean number of MPCEs in the Cyclophosphamide treated mice was above the the corresponding historical control data range, however within the assay acceptance range.

The single oral administration of 500 mg/kg bw, 1000 mg/kg bw and 2000 mg/kg body weight of tert. butylperoxy-3,5,5-trimethylhexanoate (TBPIN) did not induce biologically and statistically significant increases in the frequency of MPCEs in male mice at either 24 or 48 hours after the treatment compared to the concurrent negative (solvent) control group.

- Ratio of PCE/NCE:
The ratio of polychromatic to normochromatic cells was calculated on the basis of the number of mature cells encountered while accumulating 200 PCEs. At the 24-hour sampling the number of PCEs was decreased in the 1000 and 2000 mg/kg bw dose groups and at the 48-hour sampling in the 2000 mg/kg bw compared to the negative control group value. This effect demonstrated exposure of the bone marrow to the test item.


RESULTS OF DEFINITIVE STUDY

Clinical Signs and Mortality:

No animals died during the study. No adverse reactions to treatment were observed in the mice of the untreated, negative and positive control groups. The mice dosed at the 500 and 1000 mg/kg bw dose levels were symptom-free during the study. The male animals dosed 2000 mg/kg bw showed slight decreased in activity and piloerection at two and at five hours after the treatment and moderate decreased in activity and piloerection were observed between 3-4 and four hours after the treatment.

Conclusions:
Under the conditions of this assay the test item tert. butylperoxy-3,5,5-trimethylhexanoate (TBPIN) did not induce any statistically and biologically relevant increase in the number of micronucleated polychromatic erythrocytes at dose levels of 500, 1000 and 2000 mg/kg body weight after oral administration on one occasion in NMRI BR mice.
Executive summary:

The potential mutagenic activity of tert. butylperoxy-3,5,5-trimethylhexanoate (TBPIN) was examined in bone marrow of male NMRI BR mice. Based on a preliminary oral toxicity study doses of 500, 1000 and 2000 mg/kg bw were selected. Untreated negative (solvent) control and a positive control groups were included. Treatment was carried out with TBPIN by an oral single dosage. The test item was dissolved Sunflower oil with a constant treatment volume (10 mL/kg body weight). Cyclophosphamide dissolved in 0.9% NaCl solution (positive control) was administered once, intraperitoneally with a treatment volume of 10 mL/kg body weight. In the untreated control, low and mid dose groups the sampling from bone marrow was performed once at 24 hours after treatment and twice, at 24 and 48 hours after treatment in the high dose and the negative control groups. In animals treated with Cyclophosphamide (60 mg/kg bw.), the sampling was performed only at 24 hours post-treatment. Five animals per dose group were used on each occasion. Two thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells.

The frequencies of micronucleated polychromatic erythrocytes (MPCEs) for the untreated, negative (solvent) and positive control mice were within acceptable ranges and compatible with the historical control data for this laboratory. The positive control values showed a large, statistically significant increase over the negative control values, demonstrating the sensitivity of the test. At the 24-hour sampling the number of PCEs was decreased in the 1000 and 2000 mg/kg bw dose groups and at the 48-hour sampling in the 2000 mg/kg bw compared to the negative control group value. This effect demonstrated exposure of the bone marrow to the test item.

The single oral administration of 500 mg/kg bw, 1000 mg/kg bw and 2000 mg/kg body weight of TBPIN did not induce biologically or statistically significant increase in the frequency of MPCEs in male mice at either 24 or 48 hours after the treatment compared to the concurrent control group.

In conclusion, no biologically or statistically significant increases in the frequency of MPCEs were seen in the groups of mice treated with TBPIN to the negative control group.Thus TBPIN did not show genotoxic activity in this mouse micronucleus test.

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

Additional information

Additional information from genetic toxicity in vitro and in vivo:


Tert-Butylperoxy-3,5,5-trimethylhexanoat was tested for genetic toxicity in four in vitro tests:


The mutagenic activity of the test substance was examined in an Ames test, using a set of four histidine requiring mutants of Salmonella typhimurium (TA98, TA100, TA1535, TA1537) and an E.coli WP2 uvrA strain with addition of  liver homogenate of phenobarbital- and naphthoflavone-induced rats (S9 mix). Mutagenic effects were noted for TA98, TA100, TA1537 and E.coli WP2 uvrA with metabolic activation.


 


The test substance was further tested in a mammalian gene mutation test in V79 cells according to EU method B.17. In two independent main experiments 9.76, 39.06, 78.12, 156.25, 234.37 and 312.50 µg/mL of the test substance in DMSO were incubated. Results showed that the test substance was not mutagenic in V79 cells.


 


The test substance was tested in two in vitro mammalian chromosome aberration test studies. The potential of the test item to induce chromosome aberrations was investigated in cultured human lymphocytes. The test item was investigated in two independent experiments. Under this experimental conditions, the test item induced chromosome aberrations in cultured human lymphocytes, with and without metabolic activation (S9 mix). A second in vitro mammalian chromosome aberration test study confirmed the findings of the first study as chromosome aberration was observed with metabolic activation (S9 mix).


 


To clarify the positive response in the chromosome aberrations assays tert-Butylperoxy-3,5,5-trimethylhexanoat was tested in an in vivo micronucleus assay on mice.


 


The single oral administration of 500 mg/kg bw, 1000 mg/kg bw and 2000 mg/kg body weight did not induce biologically or statistically significant increase in the frequency of MPCEs in male mice at either 24 or 48 hours after the treatment compared to the concurrent control group. In conclusion, no biologically or statistically significant increases in the frequency of MPCEs were seen in the groups of mice treated with the test substance to the negative control group. Thus, the test substance is considered not to be clastogenic in vivo.


 


To clarify the positive results from the Ames test, an in vivo comet assay is proposed.


 


 


 

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data show positive effects both in an in vitro gene mutation study in bacteria and in an in vitro cytogenicity/chromosome abberation study in mammalian cells. A comet assay in vivo is proposed to clarify classification of the substance under Regulation (EC) No 1272/2008.