tert-butyl 3,5,5-trimethylperoxyhexanoate

Genetic toxicity: in vitro

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

Test guideline 3

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

Test material information

Constituent 1

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-trimethylhexaneperoxoate

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 / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Species / strain 2

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.

Controls

Controls 1

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)

Controls 2

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)

Controls 3

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)

Controls 4

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)

Controls 5

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 results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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

Test results 6

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

Test results 7

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

Test results 8

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

Test results 9

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

Test results 10

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).