3-(3,5-di-tert-butyl-4-hydroxyphenyl)-N-octadecylpropanamide

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:

31 January to 27 February 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study undertaken at GLP accredited laboratory to internationally accepted guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

T-1215A

IUPAC Name:

T-1215A

Constituent 2

Reference substance name:

3-(3,5-di-tert-butyl-4-hydroxyphenyl)-N-C16-18- linear alkyl-propionamide.

IUPAC Name:

3-(3,5-di-tert-butyl-4-hydroxyphenyl)-N-C16-18- linear alkyl-propionamide.

Details on test material:

- Name of test material (as cited in study report): T-1215A
- Substance type:
- Physical state: White powder
- Analytical purity: 100%
- Purity test date: 18 August 2010
- Lot/batch No.: 10180U
- Expiration date of the lot/batch: 17 March 2012
- Storage condition of test material: Room temperature

Method

Target gene:

This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient genotype (TK-/-).

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
R0; RPMI 1640, buffered with 2 mg/mL sodium bicarbonate, supplemented with 2.0 mM L-glutamine and 50 μg/mL gentamicin.
R10p; R0, supplemented with 0.1% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 10% v/v.
R30p; R0, supplemented with 0.02% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 30% v/v.

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

other: thymidine kinase deficient genotype

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

3.75, 7.5, 15, 30, 60 and 120 µg/ml

Vehicle / solvent:

- Vehicle used: Dimethyl formamide
- Justification for choice of solvent/vehicle: Test substance soluble in vehicle and showed no precipitate in the culture medium.

Controls

Controls 1

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Solvent; DMSO

Controls 2

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Solvent; DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension;

DURATION
- Exposure duration:3 and 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 7 to 14 days

NUMBER OF REPLICATIONS: 1

NUMBER OF CELLS EVALUATED: 2E03

OTHER EXAMINATIONS:
- Other: On completion of each main mutagenicity test, data were examined for cell growth parameters, cytotoxicity, plating efficiencies, spontaneous and positive control MF, and percent small colonies in positive control cultures.

Evaluation criteria:

Tests were accepted on the basis of the following criteria:

Acceptance criteria for test substance:
The highest concentration tested was one that allowed the maximum exposure up to 5000 μg/mL or 10 mM for freely soluble compounds, or the limit of toxicity (ie. relative total growth reduced to approximately 10 to 20% of the concurrent vehicle control) or the limit of solubility. For a toxic substance, at least 4 analysable concentrations should have been achieved which ideally spanned the toxicity range of 100 to 10% RTG.

Acceptance criteria for vehicle controls:
The mean vehicle control value for mutant frequency was between 50 to 170 x 10-6.

The mean cloning efficiency was between 65 to 120%.

The mean suspension growth was between 8 to 32 on Day 2 following 3 hour treatments and between 32 to 180 on Day 2 following a 24 hour treatment.

Obvious outliers were excluded. However, there were at least 2 vehicle control cultures remaining.

Acceptance criteria for positive controls:
Positive controls showed an absolute increase in mean total MF above the mean concurrent vehicle control MF of at least 300 x 10-6. At least 40% of this was due to the number of small mutant colonies.

Mean RTG’s for the positive controls were greater than 10%.

There was an absence of confounding technical problems such as contamination, excessive numbers of outliers and excessive toxicity.

There was not excessive heterogeneity between replicate cultures.

Assays that did not fulfil the required criteria were rejected and therefore are not reported. This decision was at the discretion of the Study Director.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: Preliminary toxicity test
Cells were exposed to the test substance for 3 hours in the absence and presence of S9 mix and for 24 hours in the absence of S9 mix.

For 3 hour exposures, cultures contained a total of 6 x 106 cells. The final volume of the cultures was 5 mL and the final concentration of the S9 fraction was 2% v/v, if present. For 24 hour exposures, cultures contained a total of 1.5 x 106 cells in a total volume of 5 mL. One culture was prepared for each concentration of the test substance for each test condition. Vehicle controls were tested in duplicate for each test condition. The test substance was formulated and serially diluted in the solvent. Aliquots of 50 μL of test substance dilution (at 100 times the desired final concentration) or vehicle were added to each culture prior to incubation for 3 hours (continuous shaking at 37°C) or 24 hours (static incubator, at 37°C, 5% (v/v) CO2). At the end of the 3 hour exposure period, the cells were washed once, resuspended in R10p to nominally 2 x 105 cells/mL (assuming no cell loss), incubated and sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 105 cells/mL with R10p where necessary. At the end of the 24 hour exposure period, the cells were washed once, resuspended in 5 mL R10p and counted, to ascertain treatment growth. The cultures were then diluted to
2 x 105 cells/mL with R10p as appropriate, incubated and sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 105 cells/mL with R10p where necessary.

The RSG was used to determine the concentrations of test substance used in the main test; ideally the maximum concentration should reduce RTG to approximately 10 to 20% of the concurrent vehicle control value. There was limited evidence of toxicity in the preliminary toxicity test, so the maximum concentration tested in the 3 hour exposure in the absence and presence of S9 mix were 120 μg/mL, and in the 24 hour exposure in the absence of S9 mix was 120 μg/mL. The formulations were added at 1% final volume in medium.

Remarks on result:

other: strain/cell type: forward mutation (TK-/-)

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

It was concluded that T-1215A did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.