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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Chemical structure
Reference substance name:
tert-butyl 3,5,5-trimethylperoxyhexanoate
EC Number:
236-050-7
EC Name:
tert-butyl 3,5,5-trimethylperoxyhexanoate
Cas Number:
13122-18-4
Molecular formula:
C13H26O3
IUPAC Name:
tert-butyl 3,5,5-tris(methylperoxy)hexanoate

Method

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 / strainopen allclose all
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.
Controlsopen allclose all
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.

Results and discussion

Test resultsopen allclose all
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"

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.

Applicant's summary and conclusion

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).