2,5-di-tert-butyl-p-benzoquinone

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:

19 jul 2016 5 Oct 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,5-di-tert-butyl-p-benzoquinone

EC Number:

219-552-0

EC Name:

2,5-di-tert-butyl-p-benzoquinone

Cas Number:

2460-77-7

Molecular formula:

C14H20O2

IUPAC Name:

2,5-di-tert-butyl-1,4-benzoquinone

Test material form:

solid: bulk

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 97

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain 5

Species / strain / cell type:

E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix induced by Arocolor

Test concentrations with justification for top dose:

50, 150, 500, 1500 and 5000 ug/ml

Vehicle / solvent:

Tetrahydrofuran (THF).

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-nitro-1,2-phenylene and 2-amino-anthracene

Details on test system and experimental conditions:

Salmonella typhimurium and E.coli (all strains used) were obtained from TRINOVA Bio-Chem (batch of the Salmonella typhimurium strains: TA97a: 4997D, TA98: 5011D, TA100: 4996D, TA1535: 5012D and batch of E. coli strain: 4999D) and were stored as lyophilisates in the fridge at 2-8 °C.The lyophilisates were used to prepare permanent cultures which were filled into vials and stored at < - 75 °C 8 h before the start of each experiment, an aliquot of a permanent culture per strain to be used was taken from the deep freezer to inoculate a culture vessel containing nutrient broth. After incubation overnight for 8 hours at 37 ± 1 °C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature as to prevent changes in the titre.

Rationale for test conditions:

8 h before the start of each experiment, one vial permanent culture of each strain was taken from the deep freezer and an aliquot was put into a culture flask containing nutrient broth. After incubation for 8 hours at 37 ±1 °C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature as to prevent changes in the titre.

Evaluation criteria:

The colonies were counted visually and the numbers were recorded. A spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control. The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) 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 (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.
A substance is considered to have mutagenic potential, if a reproducible increase of revertant 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.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that 2,5-Di-tert-butyl-1,4- benzoquinone is not mutagenic in the bacterial reverse mutation test (OECD 471).

Executive summary:

The study procedures were based on OECD 471 guidelines. The test item 2,5-Di-tert-butyl-1,4-benzoquinone was tested in the Salmonella typhimurium reverse mutation assay with four strains of Salmonella typhimurium (TA97a, TA98, TA100 and TA1535) and one strain of Escherichia coli (WP2) using doses 50, 150, 500, 1500 and 5000 ug/ml. The test was performed in two experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254). The results of this experiments showed that the test item 2,5-Di-tert-butyl-1,4- benzoquinone caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation.