Benzenamine, N-phenyl-, styrenated

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:

07 March 2016 - 02 June 2016 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

OECD Guidelines for Testing of Chemicals No. 476 "In Vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes”. Adopted 28 July 2015.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

In principle Guideline OECD 476 will satisfy the requirements of the following guideline: Method B17 of Commission Regulation (EC) No 440/2008 of 30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

In principle Guideline OECD 476 will satisfy the requirements of the following guideline: US EPA OPPTS 870.5300

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Issued by the Department of Health of the Government of the United Kingdom

Type of assay:

other: forward gene mutation assay in mammalian cells

Test material

Test material information

Constituent 1

Reference substance name:

Benzenamine, N-phenyl-, styrenated

EC Number:

270-485-3

EC Name:

Benzenamine, N-phenyl-, styrenated

Cas Number:

68442-68-2

Molecular formula:

not applicable (UVCB substance)

IUPAC Name:

4-(1-phenylethyl)-N-[4-(1-phenylethyl)phenyl]aniline

Test material form:

liquid: viscous

Details on test material:

- Storage Conditions: Room temperature in the dark

Method

Target gene:

hprt

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: The V79 cell stocks were obtained from Harlan CCR in 2010 and originated from Labor für Mutagenitätsprüfungen (LMP); Technical University; 64287 Darmstadt, Germany.
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. The high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50 %) make it an appropriate cell line to use for this study type. The cells have a stable karyotype with a modal chromosome number of 22.
- Methods for maintenance in cell culture if applicable: Laboratory stock cell cultures will be periodically checked for stability and absence of mycoplasma contamination. The stock of cells is stored in liquid nitrogen. For use, a sample of cells will be removed before the start of the study and grown in Eagles Minimal Essential (MEM) (supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10% foetal bovine serum (FBS)) at approximately 37 °C with 5% CO2 in humidified air.
- Modal number of chromosomes: 22
- Normal (negative control) cell cycle time: doubling time 12 - 16 h in stock cultures

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: see above
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes: Before a stock of cells is frozen for storage the number of pre-existing HPRT-deficient mutants must be reduced. The cells are cleansed of mutants by culturing in HAT medium for four days. This is MEM growth medium supplemented with Hypoxanthine (13.6 μg/mL, 100 μM). Aminopterin (0.0178 μg/mL, 0.4 μM) and Thymidine (3.85 μg/mL, 16 μM). After four days in medium containing HAT, the cells are passaged into HAT free medium and grown for four to seven days. Bulk frozen stocks of these “HAT” cleansed cells are frozen down prior to use in the mutation studies, with fresh cultures being removed from frozen before each experiment.

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Microsomal Enzyme Fraction: Phenobarbital/beta-Naphthoflavone induced rat liver S9 (Sprague-Dawley, male)

Test concentrations with justification for top dose:

The test item was considered to be an UVCB and therefore the maximum recommended dose concentration was 5000 μg/mL. Hence, the dose levels of test item used in the preliminary cytotoxicity test were 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 μg/mL. Results from the preliminary cytotoxicity test were used to select the test item dose levels for the mutagenicity experiment. The dose range of test item was 5 to 80 μg/mL in the absence of metabolic activation (S9) and 5 to 60 μg/mL in the presence of S9.
The maximum dose level selected for the main mutagenicity experiment was the lowest precipitating dose level in the absence of S9 (80 μg/mL) and in the presence of S9 the dose selection was based on the toxicity seen in the preliminary toxicity test and was 60 μg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was insoluble in culture medium at 50 mg/mL but was soluble in dimethyl sulphoxide (DMSO) at 500 mg/mL.

Controls

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: Dimethyl benzanthracene (DMBA), stereochemistry not specified

Remarks:

Solvent for positive controls: DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): Cells were seeded at 1 x 10exp7 cells/225 cm² flask approximately 24 hours being exposed to the test or control items. This was demonstrated to provide at least 20 x 10exp6 available for dosing in each flask using a parallel flask.

DURATION
- Preincubation period: 24h
- Exposure duration: 4h
- Expression time (cells in growth medium): During the 7 Day expression period the cultures were sub-cultured and maintained on days 2 to 5 to maintain logarithmic growth
- Selection time (if incubation with a selection agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): approx 14 days

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

STAIN: 10% Giemsa solution

NUMBER OF REPLICATIONS: triplicates for cloning efficiency, 10 replicates per group for mutation frequency

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Flasks were fixed with methanol and stained with Giemsa

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Rationale for test conditions:

standard requirements

Evaluation criteria:

Interpretation of Results

Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly positive if, in any of the experimental conditions examined:
i) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) The increase is considered to be concentration-related.
iii) The results are outside the range of the historical negative control data for the test item concentrations.
When all these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.

Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in all of the experimental conditions examined:
i) None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) There is no concentration related increase.
iii) The results for the test item concentrations are within the range of the historical negative control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
There is no requirement for verification of a clearly positive or negative response.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgment and/or further investigations. Performing a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing, S9 concentration, and exposure time) may be useful.

Statistics:

Statistical analysis
When there is no indication of any increases in mutant frequency at any dose level then statistical analysis may not be necessary. In all other circumstances comparisons will be made between the appropriate vehicle control value and each individual dose level, using Student’s t-test. Other statistical analysis may be used if they are considered to be appropriate.

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Remarks:

vehicle control

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test item was dosed into media.
- Effects of osmolality: osmolality did not increase by more than 50 mOsm at the dose levels investigated
- Evaporation from medium: none stated
- Water solubility:
- Precipitation: A precipitate of the test item was observed at the end of exposure at and above 78.13 μg/mL in the 4-hour exposure group in both the absence and presence of S9.
- Definition of acceptable cells for analysis:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:
Preliminary Cytotoxicity Test
A dose range of 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 μg/mL were used in the preliminary cytotoxicity test. The maximum dose tested was the maximum recommended dose level for an UVCB substance.
A precipitate of the test item was observed at the end of exposure at and above 78.13 μg/mL in the 4-hour exposure group in both the absence and presence of S9. There was modest toxicity demonstrated in the absence of S9 and there was a 57% reduction in cloning efficiency at 5000 μg/mL and a 15% reduction in cloning efficiency at the lowest precipitating dose level (78.13 μg/mL) when compared to the vehicle control. The toxicity demonstrated in the presence of S9 was much more marked and there was 43% and 98% reduction in cloning efficiency at 39.06 μg/mL and 78.13 μg/mL, respectively.
The maximum dose level selected for the main mutagenicity experiment was the lowest precipitating dose level in the absence of S9 (80 μg/mL) and in the presence of S9 the dose selection was based on the toxicity seen in the preliminary toxicity test and was 60 μg/mL.

Applicant's summary and conclusion

Conclusions:

The study was conducted under GLP according to OECD guideline 476 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the potential of Benzenamine, N-phenyl-, styrenated to induce gene mutations in V79 cells.
The test item did not induce any significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation. The test item was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.

Executive summary:

The purpose of this GLP OECD 476 guideline study is to assess the potential mutagenicity of Benzenamine, N-phenyl-, styrenated, on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line.

 

Chinese hamster (V79) cells were treated with the test item at up to seven dose levels, in duplicate, together with vehicle (dimethyl sulphoxide) and positive controls in the presence and absence of an S9 metabolic activation system.

The dose levels used in the Main Mutagenicity Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by precipitate in the absence of S9 and toxicity in the presence of S9. The dose levels selected for the Main Test were 5, 10, 20, 40, 60, 80 μg/mL over 4-hours without S9, and 5, 10, 20, 40, 45, 50, 60 μg/mL over 4-hours with S9 (2%).

The assay acceptance criteria were achieved, in both exposure groups, in terms of cloning efficiency, number of cells plated, etc. The vehicle (dimethyl sulphoxide) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus. The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolizing system.

The test item demonstrated no significant increases in mutant frequency at any test item dose level, either with or without metabolic activation. The dose range used in the absence of S9 included the lowest precipitating dose level and in the presence of S9 included the appropriate range of toxicity including one dose level that induced 90% growth inhibition.

 

Benzenamine, N-phenyl-, styrenated was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.