4,4',4''-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 September 1992 to 10 November 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to OECD test guidance in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4,4',4''-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol]

EC Number:

217-420-7

EC Name:

4,4',4''-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol]

Cas Number:

1843-03-4

Molecular formula:

C37H52O3

IUPAC Name:

4-[4,4-bis(5-tert-butyl-4-hydroxy-2-methylphenyl)butan-2-yl]-2-tert-butyl-5-methylphenol

Method

Target gene:

L5178Y TK+/- mouse lymphoma cells

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

A bank of L5178Y (-3.7.2c [TK+/-]) cells was stored in a liquid nitrogen freezer. The cell stocks have been shown to be free of mycoplasma by enzyme linked immunosorbent assay (ELISA).
The cultures were kept at 37°C under an atmosphere of 5% CO2 in air, either in a gassing incubator or in a hot room in roller bottles rotated on a roller apparatus.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

0 - 125μg/ml

Vehicle / solvent:

Dimethylsulphoxide (DMSO), CTL reference number Y00876/001 (BDH Chemicals Ltd, England), was used as the vehicle for the positive controls.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Migrated to IUCLID6: N-nitrosodimethylamine (NDMA)

Details on test system and experimental conditions:

Control Chemicals
Negative Control: Dimethylsulphoxide (DMSO), CTL reference number Y00876/001 (BDH Chemicals Ltd, England), was used as the vehicle for the positive controls. It was also tested independently as the solvent control (SC) at an equivalent volume (i.e .200μl added to 20ml cell cultures) both in the absence and presence of S9.
Positive Controls: The positive controls were ethyl methanesulphonate (EMS), CTL reference number Y01958/005 (Sigma, England); and N-nitrosodimethylamine (NDMA), CTL reference Y01468/005 (Sigma, England). EMS is a direct acting mutagen and was tested in the absence of S9 at a final dose level of 750μg/ml. NDMA, which requires metabolic activation to its reactive species, was tested in the presence of S9 at a final concentration of 600μg/ml. The shelf lives of these chemicals have not yet been defined as no loss in activity has been detected to date.

Test Methods
Preparation of Solutions of Test Sample and Positive Controls:
All stock solutions were prepared by dissolving the chemical in DMSO to produce a final concentration of 100 times that of the ultimate maximum dose level. Further appropriate serial dilutions of this main stock solution were made to provide the 100 times stock solution for subsequent dose levels.
Cell Maintenance: A bank of L5178Y (-3.7.2c [TK+/-]) cells was stored in a liquid nitrogen freezer. The cell stocks have been shown to be free of mycoplasma by enzyme linked immunosorbent assay (ELISA).
The cultures were kept at 37°C under an atmosphere of 5% CO2 in air, either in a gassing incubator or in a hot room in roller bottles rotated on a roller apparatus.
Summary of Methodology: A dose-ranging study was performed to determine concentrations of 1,1,3-Tris(2-methyl-4-hydroxy-5-tertbutylphenyl)butane to be used in the main mutation assay. Subsequently, two series of exponentially growing suspension cultures of L5178Y cells (one with, and one without S9), were treated in duplicate with concentrations of 1,1,3-Tris(2-methyl-4-hydroxy-5-tertbutylphenyl)butane or control chemical (as appropriate) for 4 hours. 1,1,3-Tris(2-methyl-4-hydroxy-5-tertbutylphenyl)butane was tested both in the presence and absence of S9 up to a maximum dose level of 125μg/ml. After removal of the treatment medium, the cells were washed and a sample diluted to determine the survival immediately after treatment. The remaining cells were then cultured to allow any induced mutants to be detected. During this expression time the growth rate was monitored and the cells subcultured daily. At the end of the 72 hour expression time, samples were grown in both selective medium (supplemented with trifluorothymidine (TFT)) and non-selective medium, and the results obtained used to determine the mutant frequency per viable cell.

Evaluation criteria:

Test data from individual experiments were considered valid if:
(a) the spontaneous mutant frequencies in the presence and absence of S9 fell within an acceptable range.
(b) the positive controls induced unequivocal positive responses. A positive response in a valid individual experiment is recorded when statistically significant, dose related increases in mutant frequency are observed across a range of toxicity. Such increases in mutant frequency are not considered indicative of a positive response if the increases occur only at doses eliciting excessive toxicity, i.e. <10% survival. These increases must also be associated with increases in the numbers of mutants over those observed with the concurrent negative control.

A negative result in a valid experiment is obtained when there is no significant dose related increase in mutant frequency.

A positive response in an individual experiment must be reproducible for the test chemical to be considered positive (i.e. mutagenic) in this assay.

Statistics:

A statistical analysis is applied at the discretion of the Study Director in consultation with the Study Statistician. The data was examined by the Study Statistician who considered that a statistical analysis was not necessary.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not specified

Untreated negative controls validity:

not applicable

Positive controls validity:

not specified

Additional information on results:

From a preliminary dose-ranging experiment, the maximum dose level of 1,1,3-Tris(2-methyl-4-4hydroxy-5-tertbutylphenyl)butane that was considered appropriate for testing was determined as 125μg/ml both with and without S9 since this dose produced significant toxicity and the next dose tested (250μg/ml) gave a visible precipitate at the end of the treatment time. In the subsequent main mutation experiments, this top dose level proved too toxic in the absence of S9 and lower dose level were used to provide an assessment over an adequate toxicity range.

Survival Data
In the first experiment, 1,1,3-Tris(2-methyl-4-hydroxy-5-tertbutylphenyl)butane was examined in the presence and absence of S9 at dose levels up to 125μg/ml, which in the presence of S9 reduced survival to 9% of the negative control value. The highest surviving dose level in the absence of S9 in this experiment was 31.3μg/ml which gave 35% survival. In the second experiment, the highest surviving dose in the absence of S9 was 22μg/ml which gave 8% survival. In the presence of S9, 1% survival was observed at the top dose of 125μg/ml with 17% survival at a dose of 70μg/ml. The third experiment was conducted in the absence of S9 only, the highest surviving dose being 38μg/ml which gave 8% survival. Dose related toxicity was observed in all experiments and an evaluation of mutant frequency was made over a toxicity range extending to approximately 10% survival both in the presence and absence of S9.

Mutation Data
1,1,3-Tris(2-methyl-4-hydroxy-5-tertbutylphenyl)butane have no reproducible dose related increases in mutant frequency and is therefore considered to have given a negative response in the assay.
The positive controls (EMS and NDMA) induced substantial increases in mutant frequency in all experiments, demonstrating that the assay was performing satisfactorily.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

SUMMARY OF DATA FOR EXPERIMENT 1

WITHOUT S9				WITH S9
Dose (μg/ml)	% Survival	Mutant Frequency (x10^-4)		Dose (μg/ml)	% Survival	Mutant Frequency (x10^-4)
1,1,3-Tris(2-methyl-4-hydroxy-5-tertbutylphenyl)butane				1,1,3-Tris(2-methyl-4-hydroxy-5-tertbutylphenyl)butane
125	0	-		125	9	2.6
62.5	0	-		62.5	84	1.0
31.3	35	1.1		31.3	81	1.0
15.6	63	0.8		15.6	96	1.1
Negative Control				Negative Control
DMSO (10μl/ml)	100	1.7		DMSO (10μl/ml)	100	1.2
Positive Control				Positive Control
EMS 750	23	22.6		NDMA 600	82	18.2

 

SUMMARY OF DATA FOR EXPERIMENT 2

WITHOUT S9				WITH S9
Dose (μg/ml)	% Survival	Mutant Frequency (x10^-4)		Dose (μg/ml)	% Survival	Mutant Frequency (x10^-4)
1,1,3-Tris(2-methyl-4-hydroxy-5-tertbutylphenyl)butane				1,1,3-Tris(2-methyl-4-hydroxy-5-tertbutylphenyl)butane
53	0	-		125	1	-
40	0	-		94	0	-
30	0	-		70	17	0.7
22	8	1.6		53	49	1.2
117	39	0.9		40	71	1.6
Negative Control				Negative Control
DMSO (10μl/ml)	100	1.0		DMSO (10μl/ml)	100	1.1
Positive Control				Positive Control
EMS 750	47	13.7		NDMA 600	77	15.3

 

SUMMARY OF DATA FOR EXPERIMENT 3

WITHOUT S9
Dose (μg/ml)	% Survival	Mutant Frequency (x10^-4)
1,1,3-Tris(2-methyl-4-hydroxy-5-tertbutylphenyl)butane
50	0	-
38	8	3.1
28	51	2.9
21	83	2.5
16	99	4.0
12	145	4.0
9	107	1.9
Negative Control
DMSO (10μl/ml)	100	3.3
Positive Control
EMS 750	34	25.9

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with & without metabolic activation

Under the conditions of this assay, 1,1,3-Tris(2-methyl-4-hydroxy-5-tertbutylphenyl)butane is non-mutagenic to L5178Y TK+/- cells as determined by selection in trifluorothymidine in both the presence and absence of an auxiliary metabolising system (S9), when tested to doses limited by the toxicity of the test material.

Executive summary:

Study conducted to OCED Guidelines for Testing of Chemicals, Second addendum August 1984. Section 4, Genetic Toxicity, 476, In Vitro Mammalian Cell Gene Mutation Tests and in compliance with the Principles of Good Laboratory Practice (GLP).

To assess the mutagenic potential of 1,1,3-Tris(2-methyl-4-hydroxy-5-tertbutylphenyl)butane to mammalian cells, L5178Y TK+/- mouse lymphoma cells were treated in vitro with various concentrations of the test sample, both in the presence and absence of an auxiliary metabolising system (S9). Mutant frequencies were assessed by cell growth in the presence of trifluorothymidine after a 72 hours expression time.

1,1,3-Tris(2-methyl-4-hydroxy-5-tertbutylphenyl)butane was tested both in the presence and absence of S9 in two independent experiments and in the absence of S9 only in a third experiment. All experiments gave negative results in the presence and absence of S9 when tested to a maximum dose of 125μg/ml.

Under the conditions of this assay, 1,1,3-Tris(2-methyl-4-hydroxy-5-tertbutylphenyl)butane is non-mutagenic to L5178Y TK+/- cells in both the presence and absence of an auxiliary metabolising system (S9) when tested to doses limited by the toxicity of the test material.