N-[(1,1,3,3-tetramethylbutyl)phenyl]naphthalen-1-amine

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:

October 02, 2012 - March 06, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes (incl. QA statement)

Remarks:

Harlan Cytotest Cell Research GmbH, In den Leppsteinswiesen 19, 64380 Rossdorf, Germany

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-[(1,1,3,3-tetramethylbutyl)phenyl]naphthalen-1-amine

EC Number:

257-406-8

EC Name:

N-[(1,1,3,3-tetramethylbutyl)phenyl]naphthalen-1-amine

Cas Number:

51772-35-1

Molecular formula:

C24H29N

IUPAC Name:

N-[(1,1,3,3-tetramethylbutyl)phenyl]naphthalen-1-amine

Details on test material:

- Physical state: solid/white
- Analytical purity: 99.0 g/100 g determined by 1H-MR spectroscopy
- Expiration date of the lot/batch: July 15, 2015
- Storage condition of test material: Room temperature; avoid temperatures > 35°C

Method

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase)

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- 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
- Periodically checked for spontaneous mutant frequency: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment I:
4 hours, without S9 mix: 13.0, 26.0, 52.0, 104.0, 208.0, (416.0) µg/ml
4 hours, with S9 mix: 13.0, 26.0, 52.0, 104.0, 208.0, (416.0) µg/ml
Experiment II:
24 hours, without S9 mix: 0.8, 1.6, 3.3, 6.5, 13.0, (26.0), (52.0), (104.0) µg/ml
4 hours, with S9 mix: (13.0), 26.0, 52.0, 104.0, 208.0, 416.0 µg/ml
numbers in parantheses: these cultures were discontinued.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: THF (tetrahydrofuran)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

without S9: EMS (0.15 mg/ml); with S9: DMBA (1.1 µg/ml)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (with and without S9), 24h (without S9)
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 18 - 20 days

SELECTION AGENT (mutation assays): 11 μg/mL 6-thioguanine
STAIN (for cytogenetic assays): 10% methylene blue in 0.01% KOH solution

NUMBER OF REPLICATIONS: two independent cultures were used

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, cell density

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation fre¬quency at least at one of the concen-trations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:

A linear regression (least squares) was performed 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. However, both, biological relevance and statistical significance was considered together.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 13.0 µg/ml and higher without S9 mix and 24 hour treatment.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Phase separation, visible to the unaided eye, was noted at 52.0 µg/mL and above in experiment I with and without metabolic activation. In experiment II phase separation occurred at the maximum concentration of 416.0 µg/mL in the presence of metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
No relevant toxic effect occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment. The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Phase separation occurred at 207.5 µg/mL and above in the presence and absence of metabolic activation following 4 and 24 hours treatment. In the pre-experiment there was no relevant shift of osmolarity and pH-value of the medium even at the maximum concentration of the test item. Based on the results of the pre-experiment, the individual concentrations of the main experiments were selected.

COMPARISON WITH HISTORICAL CONTROL DATA:
In the first experiment, culture I, the mutation frequency exceeded the historical range of solvent controls (3.4 - 36.6 mutant colonies/10^6 cells) at 13.0 and 26.0 μg/mL with metabolic activation (38.0 and 48.1 mutant colonies/10^6 cells). However, the induction factor did not exceed the threshold of three times the corresponding solvent control and statistical analysis showed that there was no dose dependent increase. Therefore, the increases were judged as biologically irrelevant.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures solely occurred in the second experiment at the highest analysable concentration of 13.0 µg/mL without metabolic activation (24 hours treatment). Exceedingly severe cytotoxic effects precluded analysis of the data at the next higher concentration of 26.0 µg/mL.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of Results

	concentration (µg/ml)	PS	S9 Mix	relative cloning efficiency I (%)	relative cell density (%)	relative cloning efficiency II (%)	mutant colonies / 10^6cells	induction factor	relative cloning efficiency I (%)	relative cell density (%)	relative cloning efficiency II (%)	mutant colonies / 10^6cells	induction factor
Experiment I / 4h treatment				culture I					culture II
solvent control			-	100.0	100.0	100.0	32.0	1.0	100.0	100.0	100.0	35.7	1.0
positive control (EMS)	150.0		-	103.2	118.1	98.9	96.5	3.0	163.0	104.2	86.9	123.0	3.4
test item	13.0		-	27.1	39.5	99.3	18.4	0.6	49.4	39.1	99.6	30.9	0.9
test item	26.0		-	52.9	44.7	97.8	1.2	0.0	66.6	54.9	91.6	16.4	0.5
test item	52.0	PS	-	58.5	53.0	111.5	12.3	0.4	63.7	51.4	91.8	15.7	0.4
test item	104.0	PS	-	61.8	66.1	104.4	24.6	0.8	73.9	77.0	88.2	18.4	0.5
test item	208.0	PS	-	82.0	101.1	109.6	11.5	0.4	90.7	78.3	92.6	17.4	0.5
test item	416.0	PS	-	87.9	culture was not continued#				94.8	culture was not continued#
solvent control			+	100.0	100.0	100.0	32.1	1.0	100.0	100.0	100.0	36.5	1.0
positive control (DMBA)	1.1		+	94.0	104.7	87.9	411.9	12.8	92.6	95.4	93.8	231.6	6.3
test item	13.0		+	103.7	103.5	101.2	38.0	1.2	95.4	89.1	115.1	14.4	0.4
test item	26.0		+	96.9	109.8	84.2	48.1	1.5	94.2	99.8	97.5	20.1	0.6
test item	52.0	PS	+	95.5	101.0	93.4	16.9	0.5	94.5	114.1	115.5	36.0	1.0
test item	104.0	PS	+	96.0	106.7	85.2	17.8	0.6	86.5	91.0	110.4	21.2	0.6
test item	208.0	PS	+	93.6	111.3	87.9	27.6	0.9	88.1	113.7	110.9	33.0	0.9
test item	416.0	PS	+	96.7	culture was not continued#				89.9	culture was not continued#
Experiment II / 24h treatment				culture I					culture II
solvent control			-	100.0	100.0	100.0	18.5	1.0	100.0	100.0	100.0	5.5	1.0
positive control (EMS)	150.0		-	99.2	121.4	91.4	400.9	21.7	95.2	93.0	64.5	370.1	67.9
test item	0.8		-	101.1	115.2	97.0	13.5	0.7	101.0	112.7	94.0	6.4	1.2
test item	1.6		-	104.9	102.2	85.8	16.6	0.9	101.1	90.6	88.9	14.2	2.6
test item	3.3		-	94.7	102.2	94.5	17.2	0.9	98.9	98.8	95.5	10.4	1.9
test item	6.5		-	89.8	118.1	89.8	10.4	0.6	68.6	89.7	88.9	9.9	1.8
test item	13.0		-	41.5	43.9	96.7	11.8	0.6	40.7	40.2	89.5	4.3	0.8
test item	26.0		-	0.0	8.6	culture was not continued##			0.0	6.2	culture was not continued##
test item	52.0	PS	-	0.0	3.5	culture was not continued##			0.0	3.5	culture was not continued##
test item	104.0	PS	-	0.0	5.8	culture was not continued##			0.0	5.3	culture was not continued##
Experiment II / 4h treatment				culture I					culture II
solvent control			+	100.0	100.0	100.0	8.9	1.0	100.0	100.0	100.0	10.8	1.0
positive control (DMBA)	1.1		+	78.0	63.7	68.9	724.0	81.2	71.4	97.4	67.9	1071.7	99.2
test item	13.0		+	103.6	culture was not continued###				98.8	culture was not continued###
test item	26.0		+	110.1	79.4	87.4	12.2	1.4	107.6	113.2	95.9	15.4	1.4
test item	52.0		+	92.6	77.7	98.4	9.0	1.0	97.3	113.6	90.1	7.3	0.7
test item	104.0		+	92.1	72.6	102.7	4.8	0.5	103.7	109.1	101.2	10.9	1.0
test item	208.0		+	96.3	81.6	98.0	9.5	1.1	108.2	118.9	95.9	13.8	1.3
test item	416.0	PS	+	95.8	81.1	84.7	11.8	1.3	103.6	113.2	90.1	9.6	0.9

PS phase separation visible at the end of treatment

# culture was not continued to avoid analysis of too many concentrations displaying phase separation

## culture was not continued due to severe cytotoxic effects

### culture was not continued as a minimum of only four analysable concentrations is required

Applicant's summary and conclusion

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells and is therefore considered to be non-mutagenic in this HPRT assay.

Executive summary:

A GLP-compliant mammalian cell mutagenicity test according to OECD guideline 476 was performed to investigate the potential of the test article to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest concentration of 3320 µg/mL applied in the pre-experiment was equal to approximately 10 mM. The concentration range of the main experiments was limited by phase separation. The test item was dissolved in THF. The tested concentrations ranged from 0.8 to 416 µg/ml.

Phase separation, visible to the unaided eye, was noted at 52.0 µg/mL and above in experiment I with and without metabolic activation. In experiment II phase separation occurred at the maximum concentration of 416.0 µg/mL in the presence of metabolic activation. Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures solely occurred in the second experiment at the highest analysable concentration of 13.0 µg/mL without metabolic activation (24 hours treatment). Exceedingly severe cytotoxic effects precluded analysis of the data at the next higher concentration of 26.0 µg/mL. No relevant and reproducible increase in mutant colony numbers/10⁶ cells was observed in the main experiments up to the maximum concentration. The induction factor did not reach or exceed the threshold of 3 times the mutation frequency of the solvent controls. In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 5.5 up to 36.5 mutants per 10⁶ cells; the range of the groups treated with the test item was from 1.2 up to 48.1 mutants per 10⁶ cells. EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. Although the mutation frequency of the EMS control in both cultures of the first experiment remained well within the historical range of positive controls, the induction factor just reached or slightly exceeded the limit of 3.0. This fact is based on a relatively high mutation frequency of both solvent controls running close to the upper limit of the historical range of solvent controls. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test article is considered to be non-mutagenic in this HPRT assay.