4-tert-butyl-2-(5-tert-butyl-2-oxo-2,3-dihydro-1-benzofuran-3-yl)phenyl 3,5-di-tert-butyl-4-hydroxybenzoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-tert-butyl-2-(5-tert-butyl-2-oxo-2,3-dihydro-1-benzofuran-3-yl)phenyl 3,5-di-tert-butyl-4-hydroxybenzoate

EC Number:

700-641-2

Cas Number:

1261240-30-5

Molecular formula:

C37H46O5

IUPAC Name:

4-tert-butyl-2-(5-tert-butyl-2-oxo-2,3-dihydro-1-benzofuran-3-yl)phenyl 3,5-di-tert-butyl-4-hydroxybenzoate

Test material form:

other: Crystalline powder

Details on test material:

CAS No.: 1261240-30-5
Major ingredients: 4-tert-butyl-2-(5-tert-butyl-2-oxo-2,3-dihydro-1-benzofuran-3-yl)phenyl 3,5-di-tert-butyl-4-hydroxybenzoate
Lot/Batch No.: s25211001
Purity of active ingredient: 99-100 %
Physical description: White crystalline powder, non-sterile and odourless
Solvent and solubility: (g in 100 ml solvent at 25 degree C)
Toluene: 25
Acetone:35
Dicholomethane: > 50
Water: < 0.2

Method

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

The test substance was exposed to the S9 mixture consisted of S9 fraction (Aroclor 1254-induced) and cofactor which was used to mimic the metabolic activation system. The S9 mixture was added at final concentration of 0.94% (v/v).

Test concentrations with justification for top dose:

5; 2.5; 1.25; 0.625; 0.313 mg/plate

Vehicle / solvent:

Acetone

Controls

Untreated negative controls:

yes

Remarks:

Sterile water

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Remarks:

The positive control reference substances were selected on the basis of the type of bacterial strains used.

Positive control substance:

other: In the absence of S9: 2-nitrofluorene (2-NF), 1 µg/plate (TA 98); Sodium azide (SA), 1 µg/plate (TA 100); Mitomycin C (MMC), 0.2 µg/plate (TA 102); SA, 1 µg/plate (TA 1535); 9-aminoacridine (9-AA), 50 µg/plate (TA 1537).

Remarks:

Postive control substance: in the presence of S9: 2-aminoanthracene (2-AA), 1 µg/plate (TA 98); Benzo[a]pyrene (BP), 1 µg/plate (TA 100); 2-AA, 5 µg/plate (TA 102); 2-AA, 5 µg/plate (TA 1535); 2-AA, 5 µg/plate (TA 1537).

Evaluation criteria:

(1) The raw data of revertant colony values were represented with Mean+/- S.D.
(2) Cell toxicity determination
A cytotoxic effect was concluded when a decrease in revertant colonies over the negative/vehicle control was lower than 0.5-fold, loss of bacterial lawn, or pin colony appeared. Plates would be labeled and excluded from statistics while cytotoxic efect occurred.
(3) An increase in revertants over the negative control would be as the cut-off between a mutagenic and non-mutagenic response:
TA98, TA100 and TA 102: more than two-fold increase in revertants over the negative/vehicle control, then the test substance would be considered as a potential mutagen. TA 1535 and TA 1537: more than three-fold increase in revertants over the negative/vehicle control, then the test substance would be considered as a potential mutagen.
(4) If the test substance was considered as a potential mutagen, the raw data would be further analyzed by ANOVA to evaluate the difference between negative/vehicle control group and test substance groups, and p < 0.05 indicates the significant difference.
(5) If the data showed statistically significant, then to evaluate the dose-related response in the numbers of revertant colonies on the test substance groups as compared with the vehicle control group. Once dose-related response was confirmed, the test substance would be considered as a mutagen.
Strain: TA98, TA100, TA102; Revertants number: two-fold increase over the negative/vehicle control; Dose-dependent response: yes/no; Result: mutagenic/non-mutagenic.
Strain: TA1535, TA1537; Revertants number: trhee-fold increase over the negative/vehicle control; Dose-dependent response: yes/no; Result: mutagenic/non-mutagenic.

Results and discussion

Test results

Test results 1

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

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 3

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

(1) The genotyping of Salmonella typhimurium strains

The genotypes of five Salmonella typhimurium strains (TA98, TA100, TA102, TA1535 and TA1537) for this study swere identified. The result was organized in table 1 and Appendix D-1 of the report attached. HTe historic control data of bacterial reverse mutation test was listed in Appendix C of the report attached.

(2) Result of Dose Rang Finding Test in TA100

Five doses (5,2.5,1.25,0.625 and 0.313 mg/plate) of test substance were used, and no cytotoxic and mutagenic effects occurred. Based on the result, 5 mg/plate of test substance was determined as the highest dose for five strains bacterial reverse mutation test. All the raw data were organized in Table 2 and Appendix D-2 of the report attached.

(3) Result of Bacterial Reverse Mutation Test (5 strains)

Based on the result of dose range finding test, the concentration of 5 mg/plate of test substance was used as the highest dose and remaining four doses 2.5, 1.25, 0.625 and 0.313 mg/plate were used for bacterial reverse mutation test in five Salmonella typhimurium strains. This testing system was validated and authentic. The testing result organized in Table 3 and Appendix D-3 of the report attached. The numbers of revertant colony in negative control groups of each strain were in the range of historic control data. The revertant colonies in positive control group were more than two times (in TA98, TA100, and TA102) and three times (in TA1535 and TA1537) over negative control groups. No significant increase in the nubmers of revertant colonies was observed at all concentrations of testing strains in both presence and absence of S9 metabolic mixture, indicated the test substance did not present genotoxic effect.

Applicant's summary and conclusion

Conclusions:

negative with metabolic activation
negative without metabolic activation

According to the numbers of revertant colonies at all the testing conditions of the five strains used in this study (i.e. TA98, TA100, TA102, TA1535 and TA1537), the test substance did not present genotoxic effect at all concentrations of testing strains in the condition of both presence and absence of S9 metabolic mixture. In conclusion, test substance was non-genotoxic in the testing system applied in this study.