1-[[2-methyl-4-[(2-methylphenyl)azo]phenyl]azo]-N-tridecylnaphthalen-2-amine

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:

6.9.2012 - 10.5.2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

(see Overall remarks)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-(2-ethylhexyl)-1-[[2-methyl-4-[(2-methylphenyl)azo]phenyl]azo]naphthalen-1-amine

EC Number:

260-124-8

EC Name:

N-(2-ethylhexyl)-1-[[2-methyl-4-[(2-methylphenyl)azo]phenyl]azo]naphthalen-1-amine

Cas Number:

56358-09-9

Molecular formula:

C32H37N5

IUPAC Name:

N-(2-ethylhexyl)-1-({2-methyl-4-[(2-methylphenyl)diazenyl]phenyl}diazenyl)-1,2-dihydronaphthalen-2-amine

Test material form:

liquid: viscous

Details on test material:

- Name of test material (as cited in study report): Solvent Red 19E
- Physical state: dark viscous liquid/borderline waxy solid
- Analytical purity: 90% (w/w)
- Impurities (identity and concentrations): Solvent Red24 (CAS 85-83-6) 2% (w/w)
- Lot/batch No.: S2409
- Storage condition of test material: At room temperature, store in properly designed tanks or approved tightly closed supplied containers in cool, well-ventilated situations away from sources of ignition. Protect from static discharges.

Method

Target gene:

thymidine kinase gene (TK+/-)

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ATCC (American Type Culture Collection), 0801 University Blvd., Manassas, VA 20110-2209, USA
- Methods for maintenance in cell culture if applicable: Master stocks were maintained in liquid nitrogen.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Cells were maintained in RPMI 1640 medium1 supplemented with 0.05% Pluronic® F682, 2 mM L-glutamine3, 220 μg/mL sodium pyruvate, 100 μg/mL gentamycin4, 2.5 μg/mL fungizone5 and foetal bovine serum6 (10% by volume), hereinafter referred to as growth medium. Treatment medium was growth medium without sodium pyruvate, gentamycin and fungizone. Cleansing medium used for reducing the spontaneous frequency of TK-/- mutants prior to experimental studies consists of growth medium supplemented with approximately 4.0 x 10-5 M thymidine, 1.2 x 10-4 M hypoxanthine, 3.3 x 10-5 M glycine and 7.2 x 10-7 M methotrexate. Recovery medium is similar to cleansing medium, except that the methotrexate component is removed. Selection medium is growth medium that contains 3 μg/mL of TFT.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes.
- Periodically checked for karyotype stability: Yes
- Periodically 'cleansed' against high spontaneous background: Yes. To reduce the background mutant frequency (spontaneous frequency) of TK-/- mutants to a level as low as possible, cell cultures were exposed to conditions that select against the TK-/- phenotype (exposure to aminopterin or methotrexate). Cell cultures were maintained in cleansing medium for one day, placed in recovery medium for one day and then returned to normal growth medium for three to eight days before use.

Metabolic activation:

with and without

Metabolic activation system:

S9 liver postmitochondrial fraction

Test concentrations with justification for top dose:

15.63, 31.3, 62.5, 125, 250 and 500 µg in preliminary cytotoxicity experiment
15.63, 31.3, 62.5, 25, 125 and 250 µg Solvent Red19E/mL medium in the mutagenicity tests

A preliminary cytotoxicity experiment was performed to establish an appropriate concentration range for the mutation experiment.
A range of concentrations of 15.63, 31.3, 62.5, 125, 250, and 500 μg/mL were tested for cytotoxicity. After an exposure time of 3 hours at approx. 37°C on a roller drum at 10 - 15 rpm, the cells were washed and re-suspended in growth medium. The cells were then adjusted to 8 cells/mL and for each concentration 0.2 mL was plated into 32 microtiter wells (average 1.6 cells/well). The plates were incubated at 37°C in a humidified incubator with 5 % CO2 in air for 7 days. Wells containing viable clones were identified under a microscope and counted.
Preliminary cytotoxicity information was used to select concentration levels for the mutation assay using the following criteria:
At least four analysable concentrations were used. Where there is cytotoxicity, these concentrations cover a range from the maximum to little or no toxicity and are separated by no more than a factor between 2 and √10. If the maximum concentration is based on cytotoxicity then it should result in approximately 10 - 20% (but not less than 10 %) relative survival (relative cloning efficiency) or relative total growth.
The objective is to carry at least 5 concentrations through the entire experiment.
Based on the results of the preliminary study five concentrations of 15.63, 31.3, 62.5, 125 and 250 μg/mL medium were employed in the mutagenicity tests.
The lowest separation factor of 2 as recommended by the guidelines was used. No increase in the mutant frequency was observed. Hence, it was considered acceptable not to add any further lower concentrations, as these additional lower concentrations would provide no further information.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: As recommended by the guidelines at least 1E+06 cells were suspended in treatment medium and diluted to 5E+05 cells/mL.
Fresh preparations of the test and reference item solutions were prepared on each day of biological testing.

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

3-MC

Positive control substance:

3-methylcholanthrene

Remarks:

with metabolic activation

Controls 2

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

MMS

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hrs (with and without metabolic activation), 24 hrs (without metabolic activation)
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): 11-14 days

SELECTION AGENT (mutation assays): TFT (trifluorothymidine)

NUMBER OF REPLICATIONS: 1 (without metabolic activation), 2 (with metabolic activation)

DETERMINATION OF CYTOTOXICITY
- Method: relative survival (relative cloning efficiency)

- OTHER:
Assay without metabolic activation
The assay procedure used is based on that reported by COLE et al. (1990). Single cultures were used for each test item concentration level and reference item. The results from the initial mutation assay were confirmed by performing an independent repeat mutation assay.
The cells for the first and second experiments were obtained from logarithmically growing laboratory stock cultures of 9.7E+06 or 4.35E+06 cells/mL, respectively, and were seeded into a series of tubes, diluted to 5E+05 cells/mL per tube. The cells were pelleted by centrifugation, the culture medium was removed, and the cells were re-suspended in treatment medium that contained 5% heat inactivated foetal bovine serum and the corresponding concentration of test substance. The dosed tubes were closed, vortexed and placed on a roller drum at approx. 37 °C at 10 - 15 rpm for an exposure period of 3 hours.
Thereafter the cells were washed and re-suspended in growth medium.
Cell densities were adjusted to 2E+05/mL and the cells were plated for survival and incubated for the expression period in parallel, i.e. an aliquot of the cells was diluted to 8 cells/mL and 0.2 mL of each culture were placed in two 96 well microtiter plates (^ 192 wells, averaging 1.6 cells/well) and incubated for 1 week (plating efficiency step 1) whereas the rest of the cells was incubated for 2 days for the expression period.
The cells for the plating of survival were counted after 1 week and the number of viable clones was recorded. The cells incubated for the expression period were maintained below 106 cells per mL and a minimum of 4 concentration levels plus positive and negative control was selected for 5-trifluoro-thymidine (TFT) resistance. At the end of the second expression period the selected cultures were diluted to 1E+04 cells/mL and plated for survival (plating efficiency step 2) and TFT resistance in parallel (plating efficiency for TFT resistance). The plating for survival was identical to the above described method (plating efficiency step 1 in 192 wells with average 1.6 cells/well). For the plating for TFT resistance 3 μg/mL TFT (final concentration) was added to the cultures and 0.2 mL of each suspensions placed into four 96-well microtiter plates (^ 384 wells, averaging 2E+03 cells/well). The plates were incubated for 11 to 14 days and wells containing clones were identified microscopically and counted.
In addition, the number of large and small colonies was recorded with an automated colony counter that can detect colony diameters equal to or greater than 0.2 to 0.3 mm. Large colonies are defined as ≥ 1/4 and small colonies < 1/4 of the well diameter of 6 mm.

Assay with metabolic activation
The activation assay is often run concurrently with the non-activation assay; however, it is an independent assay performed with its own set of solvent and positive controls. In this assay the above-described activation system was added to the treatment medium together with the test item. See section 5.4 S9 metabolic activation system.
Repeat experiment

The non-activation and activation experiment were repeated in an independent experiment. An exposure time of 24 hours was used for the repeat experiment without metabolic activation (non-activation experiment). The exposure time was again 3 hours for the activation experiment. The experiments were conducted with the same concentrations as in the first experiment.

Evaluation criteria:

An assay is considered acceptable if all of the criteria given below are satisfied:
a) The mutant frequency in the negative control falls within the normal range (historical mean value).
b) The plating efficiency (Plating efficiency step 1 and step 2) of the negative control is ≥ 50%.
c) At least one concentration of the positive control induces a clear increase in the mutant frequency (the mutant frequency of the positive control is ≥ 2 times the historical mean value of the negative control) with and without S9.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Solvent Red 19E was assayed in a gene mutation assay in cultured mammalian cells (L5178Y TK+/-) both in the presence and absence of metabolic activation by a liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced rats. The test was carried out employing two exposure times without S9 mix: 3 and 24 hours, and one exposure time with S9 mix: 3 hours; the experiment with S9 mix was carried out in two independent assays.
In the preliminary experiment without and with metabolic activation (3-h exposure) test item precipitation was noted in the experiments without and with metabolic activation at concentrations of 250 and 500 μg Solvent Red 19E/mL. No signs of cytotoxicity (decreased survival) were noted in the absence and in the presence of metabolic activation up to the top concentration of 500 μg/mL.
Hence, in the main study the concentration-range of 15.63 to 250 μg/mL was used in the experiments without and with metabolic activation.
Methylmethanesulfonate (at 10 or 15 μL/mL) was employed as a positive control in the absence of exogenous metabolic activation and 3-Methylcholanthrene (at 2.5 or 4.0 μg/mL) in the presence of exogenous metabolic activation.
In the main study test item precipitation was noted in the experiments without and with metabolic activation at the top concentration of 250 μg Solvent Red 19E/mL. No signs of cytotoxicity (decreased survival) were noted in the absence and presence of metabolic activation up to the top concentration of 250 μg/mL.
The values of mutation frequencies of the negative controls ranged from 57.58 to 100.07 per 1E+06 clonable cells in the experiments without metabolic activation and from 49.79 to 87.75 per 1E+06 clonable cells in the experiments with metabolic activation and, hence, were well within the historical data-range.
The mutation frequencies of the cultures treated with Solvent Red 19E ranged from 63.98 to 82.89 per 1E+06 clonable cells (3 hours exposure) and from 63.02 to 110.56 per 1E+06 clonable cells (24 hours exposure) in the experiments without metabolic activation and from 66.12 to 77.26 per 1E+06 clonable cells (3 hours exposure, first assay) and from 80.24 to 93.79 per 1E+06 clonable cells (3 hours exposure, second assay) in the experiments with metabolic activation. These results were within the range of the negative control values and the normal range of 50 to 170 mutants per 1E+06 viable cells and, hence, no mutagenicity was observed according to the criteria for assay evaluation.
In addition, no change was observed in the ratio of small to large mutant colonies, ranging from 0.63 to 1.22 for Solvent Red 19E treated cells and from 0.59 to 1.08 for the negative controls.
The plating efficiency (PE step 1 and step 2) of the negative control was ≥ 50 %, the mean cloning efficiencies (CE) within the range of 65 % to 120 % two days after treatment, and the mean suspension growth (SG) within the range of 8 to 32 following 3-hour treatments and between 32 and 180 following 24-hour treatments. Hence, the acceptance criteria described in chapter 6.5 were met.
The positive controls Methylmethanesulfonate (MMS) and 3-Methylcholanthrene (3-MC) caused pronounced increases in the mutation frequency ranging from 1945.49 to 2399.89 per 1E+06 clonable cells in the case of MMS and ranging from 1566.03 to 1891.76 per 1E+06 clonable cells in the case of 3-MC.
In addition, the colony size ratio was moderately shifted towards an increase in small colonies, ranging from 1.71 to 2.40 in the case of MMS.
As the absolute increase in the mean total mutation frequency of at least 300E-06 (at least 40 % small colony MF) and the mean relative total growth (RTG) for the positive controls was greater than 10 %, the acceptance criteria were met.

Remarks on result:

other: strain/cell type: L5178Y TK+/-

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Negative with and without metabolic activation.

Executive summary:

The test material was assessed for genotoxicity in cultured mammalian cells (L5178Y TK +/ ) both in the presence and absence of metabolic activation according to OECD Test Guideline 476 and EC No. 440/2008 B.17: Mutagenicity in vitro mammalian cell gene test.

Solvent Red 19E was completely dissolved in dimethylsulfoxide (DMSO). The vehicle DMSO served as the negative control.

In the preliminary experiment without and with metabolic activation (24-hour or 3-h exposure) test item precipitation was noted in the experiments without and with metabolic activation at concentrations of 250 and 500 µg Solvent Red 19E/mL. No signs of cytotoxicity (decreased survival) were noted in the absence and presence of metabolic activation up to the top concentration of 500 µg/mL.

Hence, in the main study the concentration-range of 15.63 to 250 µg/mL was used in the experiments without and with metabolic activation.

Methylmethanesulfonate (at 10 or 15 µL/mL) was employed as a positive control in the absence of exogenous metabolic activation and 3-Methylcholanthrene (at 2.5 or 4.0 µg/mL) in the presence of exogenous metabolic activation.

In the main study test item precipitation was noted in the experiments without and with metabolic activation at the top concentration of 250 µg Solvent Red 19E/mL. No signs of cytotoxicity (decreased survival) were noted in the absence and presence of metabolic activation up to the top concentration of 250 µg/mL.

The values of mutation frequencies of the negative controls ranged from 57.58 to 100.07 per 106 clonable cells in the experiments without metabolic activation and from 49.79 to 87.75 per 106 clonable cells in the experiments with metabolic activation and, hence, were well within the historical data-range.

The mutation frequencies of the cultures treated with Solvent Red 19E ranged from 63.98 to 82.89 per 106 clonable cells (3 hours exposure) and from 63.02 to 110.56 per 106 clonable cells (24 hours exposure) in the experiments without metabolic activation and from 66.12 to 77.26 per 106 clonable cells (3 hours exposure, first assay) and from 80.24 to 93.79 per 106 clonable cells (3 hours exposure, second assay) in the experiments with metabolic activation. These results were within the range of the negative control values and the normal range of 50 to 170 mutants per 106 viable cells and, hence, no mutagenicity was observed according to the criteria for assay evaluation.

In addition, no change was observed in the ratio of small to large mutant colonies, ranging from 0.63 to 1.22 for Solvent Red 19E treated cells and from 0.59 to 1.08 for the negative controls.