2-(1,1-dimethylethyl)-6-[[3-(1,1-dimethylethyl)-2-hydroxy-5-methylphenyl]methyl]-4-methylphenyl acrylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Feb - 25 Jul 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted Jul 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

other: in vitro gene mutation study in mammalian cells

Test material

Test material information

Constituent 1

Reference substance name:

2-(1,1-dimethylethyl)-6-[[3-(1,1-dimethylethyl)-2-hydroxy-5-methylphenyl]methyl]-4-methylphenyl acrylate

EC Number:

262-634-6

EC Name:

2-(1,1-dimethylethyl)-6-[[3-(1,1-dimethylethyl)-2-hydroxy-5-methylphenyl]methyl]-4-methylphenyl acrylate

Cas Number:

61167-58-6

Molecular formula:

C26H34O3

IUPAC Name:

2-tert-butyl-6-[(3-tert-butyl-2-hydroxy-5-methylphenyl)methyl]-4-methylphenyl prop-2-enoate

Method

Target gene:

HPRT locus

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Laboratory for Mutagenicity Testing, Technical University, Darmstadt, Germany
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years.
- Methods for maintenance in cell culture: Thawed stock cultures were propagated at 37 °C with 1.5% carbon dioxide in 76 cm² plastic flasks and sub-cultured once or twice weekly.
- Modal number of chromosomes: 22
- Normal cell cycle time: 12 - 16 h

MEDIA USED
- Type and identity of media: MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1%).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: Yes

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone

Test concentrations with justification for top dose:

Pre-experiment for toxicity:
With and without S9 mix: 7.8, 15.6, 31.3, 62.5, 125, 250, 500 and 1000 µg/mL (4 h)

The following concentrations were selected in the main experiments for the mutagenicity testing:
Experiment I
Without S9 mix: 0.98, 1.96, 3.91, 7.83 µg/mL (4 h)
With S9 mix: 0.98, 1.96, 3.91, 7.83, 15.65 µg/mL (4 h)

Experiment II (repeat experiment)
With S9 mix: 0.98, 1.96, 3.91, 7.83, 15.65 µg/mL (4 h)

Experiment III
Without S9 mix: 0.98, 1.96, 3.91, 7.83 µg/mL (4 h)
With S9 mix: 0.98, 1.96, 3.91, 7.83, 15.65 µg/mL (4h)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures on request of the sponsor.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: Approximately 1.2 x 10E07 cells/mL

DURATION
- Exposure duration: 4 h with and without metabolic activation
- Expression time (cells in growth medium): Approximately 7 days
- Selection time (if incubation with a selection agent): About 8 days

SELECTION AGENT (mutation assays): 11 µg/mL 6-thioguanine

STAIN: 10% methylene blue in 0.01% KOH solution

NUMBER OF REPLICATIONS: Duplicates each in 3 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test item is classified as clearly positive if it induces a significant and concentration-related increase of the mutant frequency exceeding the historical solvent control range.
When a test item cannot be classified as clearly positive, additional experiments will be performed to establish the biological relevance of the result.
A test item producing a reproducible concentration-related increase of the mutant frequency above the historical solvent control range is considered to be mutagenic in this system.
A test item producing no reproducible concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.

Statistics:

A linear regression was performed using a validated test script of "R", a language and environment for statistical computing and graphics (p<0.05), to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
A t-Test was performed using a validated test script of “R”, a language and environment for statistical computing and graphics, whenever the mutation frequency at a test point exceeded the 95% confidence interval. Again a t-test is judged as significant if the p-value (probability value) is below 0.05.
However, both, biological relevance and statistical significance were considered together.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I: ≥ 3.9 µg/mL without metabolic activation; Experiment III: ≥ 3.9 µg/mL with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH-value was determined in culture medium of the solvent control and at the maximum concentration of the pre-experiment without metabolic activation and was 7.51 and 7.52, respectively.
- Effects of osmolality: The osmolarity [mOsm] was determined in culture medium of the solvent control and at the maximum concentration of the pre-experiment without metabolic activation and was 399 and 381, respectively.
- Precipitation: Precipitation was observed macroscopically and microscopically at the end of treatment (4 h) prior to the removal of the test substance at 15.6 μg/mL in the absence and presence of metabolic activation.

RANGE-FINDING/SCREENING STUDIES: A pre-test was performed in order to determine the toxicity of the test substance. The pre-experiment was performed in the presence and absence (4 h treatment) of metabolic activation. The test substance was tested at concentrations between 7.8 μg/mL and 1000.0 μg/mL. The highest concentration of the pre-experiment was chosen due to precipitation observed in the pre-test on solubility. Relevant cytotoxic effects indicated by a relative cloning efficiency of 50% or below were observed at 125.0 μg/mL in the presence and absence of metabolic activation following 4 hours treatment.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The positive control values were within the range of the historical control data (please refer to Table 1 under "any other information on material and methods incl. tables").
- Negative (solvent/vehicle) historical control data: The negative and solvent control values were within the range of the historical control data (please refer to Table 1 under "any other information on material and methods incl. tables").

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the main experiments.
In experiment I, the 95% confidence interval was exceeded at 3.91 μg/mL in culture II in the absence of metabolic activation (please refer to Table 2 under "any other information on results incl. tables"). In experiment II, the 95% confidence interval was very slightly exceeded at 7.83 μg/mL and 15.65 μg/mL in culture I (please refer to Table 3 under "any other information on results incl. tables").
A t-test evaluating the data of both parallel cultures at the data points exceeding the 95% confidence interval showed only a significant response in experiment I at 3.91 μg/mL without metabolic activation. The other data points exceeding the 95% confidence interval at 7.83 μg/mL and 15.65 μg/mL in experiment II showed no significant response in a t-test. Since the significant difference was only detected at an intermediate concentration and no dose dependent trend was found no biological relvance can be stated for this effect.

Any other information on results incl. tables

Table 1: Toxicity data

Concentration [µg/mL]	Cloning efficiency [%]
	- S9	+ S9
0 (DMSO)	100	100
7.8	101.3	90.6
15.6 P	94.4	91.0
31.3 P	80.0	80.3
62.5 P	61.6	56.7
125 P	34.4	28.8
250 P	20.6	17.0
500 P	13.3	9.0
1000 P	47.0	19.6

Table 2: Summary of Experiment I (4 h, without metabolic activation)

Concentration [µg/mL]	Cloning efficiency [%] Culture I	Mutation colonies per 10E06 cells Culture I	Cloning efficiency [%] Culture II	Mutation colonies per 10E06 cells Culture II	95% confidence intervall
Medium	154.1	21.1	76.2	19.6	1.7 - 30.2
DMSO	100	13.4	100	11.2	1.7 - 30.2
0.98	80.2	14.5	81.1	20.6	1.7 - 30.2
1.96	68.9	29.1	77.5	21.4	1.7 - 30.2
3.91	24.0	16.1	28.2	55.6	1.7 - 30.2
7.83	11.1	16.0	9.8	19.7	1.7 - 30.2
15.65 P	5.4	n.r.	5.3	n.r.	1.7 - 30.2
31.3 P	0.7	#	0.0	#	1.7 - 30.2
EMS	91.3	262.0	74.3	851.6	1.7 - 30.2

P: Precipitation (visible at the beginning and at the end of treatment)

# culture was not continued due to exceedingly severe cytotoxic effects

n.r.: not reported, due to unacceptable cytotoxicity

EMS: ethylmethane sulfonate

Table 3: Summary of Experiment II (4 h, with metabolic activation)

Concentration [µg/mL]	Cloning efficiency [%] Culture I	Mutation colonies per 10E06 cells Culture I	Cloning efficiency [%] Culture II	Mutation colonies per 10E06 cells Culture II	95% confidence intervall
Medium	114.2	22.5	77.3	26.6	2.0 – 29.4
DMSO	100.0	17.4	100.0	29.3	2.0 – 29.4
0.98	104.7	28.4	88.7	15.1	2.0 – 29.4
1.96	124.6	16.7	79.5	18.9	2.0 – 29.4
3.91	111.6	22.5	72.3	21.1	2.0 – 29.4
7.83	91.1	29.6	71.3	26.8	2.0 – 29.4
15.65 P	111.0	31.2	71.0	17.1	2.0 – 29.4
31.3 P	102.2	##	65.4	##	2.0 – 29.4
DMBA	93.2	187.8	57.0	152.3	2.0 – 29.4

P: Precipitation (visible at the beginning and at the end of treatment)

## culture was not continued to avoid analysis of too many precipitating concentrations

DMBA: 7,12-dimethylbenz(a)anthracene

Table 4: Summary of Experiment III (4 h, with and without metabolic activation)

Concentration [µg/mL]	Cloning efficiency [%] Culture I	Mutation colonies per 10E06 cells Culture I	Cloning efficiency [%] Culture II	Mutation colonies per 10E06 cells Culture II	95% confidence intervall
without metabolic activation
Medium	101.1	11.5	122.2	12.4	1.7 - 30.2
DMSO	100.0	16.1	100.0	17.9	1.7 - 30.2
0.98	72.1	14.3	107.1	13.5	1.7 - 30.2
1.96	48.1	29.3	91.7	17.1	1.7 - 30.2
3.91	25.8	16.0	43.2	21.7	1.7 - 30.2
7.83	17.4	24.0	33.0	17.6	1.7 - 30.2
15.65 P	3.6	n.r.	7.7	n.r.	1.7 - 30.2
31.3 P	1.3	#	4.5	#	1.7 - 30.2
EMS	81.2	133.6	108.1	159.4	1.7 - 30.2
with metabolic activation
Medium	117.9	13.2	142.2	20.5	2.0 – 29.4
DMSO	100.0	33.4	100.0	15.2	2.0 – 29.4
0.98	116.9	17.6	138.2	25.7	2.0 – 29.4
1.96	117.1	6.2	99.8	18.1	2.0 – 29.4
3.91	86.9	16.9	147.0	26.8	2.0 – 29.4
7.83	108.8	25.1	132.4	25.0	2.0 – 29.4
15.65 P	98.5	25.3	94.7	19.3	2.0 – 29.4
31.3 P	15.0	##	9.1	##	2.0 – 29.4
DMBA	97.5	216.6	118.8	152.6	2.0 – 29.4

P: Precipitation (visible at the beginning and at the end of treatment)

# culture was not continued due to exceedingly severe cytotoxic effects

## culture was not continued to avoid analysis of too many precipitating concentrations

n.r.: not reported, due to unacceptable cytotoxicity

EMS: ethylmethane sulfonate

DMBA: 7,12-dimethylbenz(a)anthracene

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative