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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Bacterial Reverse Mutation Test (B.13/14, GLP), with and without metabolic activation, S.typhimurium TA98, TA100, TA 1535 and TA1537, E.coli WP2 uvrA: mutagenic for tester strains S. typhimurium TA 100, TA 98 and TA 1535 with metabolic activation, non-mutagenic for all the used tester strains without metabolic activation as well as for S. typhimurium TA 1537 and E. coli WP2 uvrA with metabolic activation.

- Micronucleus Test in cultured CHO Cells in vitro (OECD 487, GLP), with and without metabolic activation, Chinese hamster ovary (CHO-K1) cells: no indications of genotoxic properties.

- Mouse Lymphoma Assay forward Mutation assay in vitro (B.17, GLP), with and without metabolic activation, L5178Y (TK+/-) mouse lymphoma cells: neither induce mutations nor have any chromosomal aberration potential.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30.8.2012 - 9.11.2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Gene for synthesis of histidine or tryptophan

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

- Genotypes of each strain were controlled (plasmid pKM 101 – ampicillin resistance, uvr mutation, rfa mutation, his/trp mutation – spontaneous reversions).

Additional strain / cell type characteristics:

other: histidine dependent strain

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Genotypes of each strain were controlled (plasmid pKM 101 – ampicillin resistance, uvr mutation, rfa mutation, his/trp mutation – spontaneous reversions).

Additional strain / cell type characteristics:

other: tryptophan dependent strain

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of rat liver homogenate and mixture of cofactors

Test concentrations with justification for top dose:

I. experiment - 15, 50, 150, 500 and 1500 µg per plate (with and without metabolic activation)
II. experiment - 15, 50, 150, 500 and 1500 µg per plate (without metabolic activation), 50, 100, 250, 500, 1000 µg per plate (with metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamine, 2-aminofluorene, 2-aminoanthracene, N-methyl-N´-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

METHOD OF APPLICATION: Plate incorporation

DURATION
- Exposure duration: 48 – 72 h

SELECTION AGENT (mutation assays): Tryptophan and histidine

NUMBER OF REPLICATIONS: Each test concentration was assessed in triplicate, except for the toxicity test where duplicates were performed.

DETERMINATION OF CYTOTOXICITY
- Method: Relative total growth

- OTHER:
Test Procedure
100 µL of test substance of required concentration, 100 µL of 16 – 18 h culture of tester strain, 0.5 mL relevant buffer and 30 µL of S9 post-mitochondrial fraction (in case of test with metabolic activation) were added to the 2 mL of molten top agar with trace of histidine or tryptophan and kept in a test tube at 45 ± 3 °C. After shaking the mixture was poured into minimal glucose agar plate. After incubation for 48 – 72 hours at 37 ± 1 °C the number of revertant colonies on the plate were counted manually with the exception of positive controls which were counted by an AccuCount 1000. Each experiment was repeated.

Selection of Doses / Toxicity
The test substance was dissolved in the maximum recommended dose (5000 µg/0.1 mL) in DMSO. It did not succeed. The maximum of the test substance dissolved in DMSO was less than 1000 µg/0.1 mL. Toxicity was tested in concentration series 10 – 1000 µg/plate, completed by the dose of 5000 µg per plate (nominal values, undissolved). This concentration series was then tested for toxicity in strain TA 100 without metabolic activation.
Turbidity appeared from the dose 200 µg per plate and the test substance made a homogenous film on the surface of the top agar. The test substance was not toxic at any dose and evaluation was possible in all doses.
The test substance is soluble in acetone and in small amounts is comparable with the test so the toxicity test was repeated with the test substance dissolved in this solvent. The test substance was dissolved in acetone at the concentration 100 mg/mL (10 mg/0.1 mL) and a concentration series (20 – 10 000 µg/0.1 mL) were made by dilution. From these applications forms, 50 µL was dosed per plate (doses 10 – 5000 µg/plate). After adding to top agar, the test substance cleaved at tube walls starting with concentration of 1000 µg/plate. On plates, the test substance was observable as a black non-homogeously spread bubbles and mist. In addition, acetone itself decreased the number of revertants with regard to spontaneous reversion. Even if the test substance was not toxic at any dose, testing in acetone was disclaimed and in the end the test substance was diluted in DMSO.
Tee dose of 1500 µg/plate was therefore used as maximum for the first mutagenicity experiments. The maximum dose was diluted accordingly to five different analysable concentrations with approximate half-log intervals between test points. There were no problems with toxicity or with evaluation. In some experiments mutagenicity was decreased or stagnated at the highest dose. The second experiments with metabolic activation were modified to obtaining better dose-response dependence from 50 to 1000 µg/plate.
Fresh solutions of test substance were prepared before each experiment. All concentrations of the test substance solution were dosed in the volume of 0.1 mL/plate.

Evaluation criteria:

The main criterion for evaluation of results was modified two-fold increase rule, which is compatible with the application of statistical methods. After this rule the result is positive, if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc is reached.

An increase is considered “biologically relevant”:
if the number of reversions is at least twice as high as that in the solvent control for the strains having spontaneous reversion > 10;
if the number of reversions is at least three times as high as that in the solvent control for the strains having spontaneous reversion ≤ 10;
A test substance producing neither a dose-related increase in the number of revertants not a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
According to OECD Test Guideline 471, the biological relevant of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.


References:
1. Maron D. M., Ames B. N. (1983): Revised methods for the Salmonella mutagenicity test, Mutat. Res. 113, 173 - 215
2. Dunkel V. C., Chu K.C. (1980): Evaluation of methods for analysis of microbial mutagenicity assays, in The Predictive Value of Short-Term Screening Tests in Carcinogenicity Evaluation, Elsevier North-Holland Biomedical Press, 231 - 240

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The results obtained in most experiments without metabolic activation did not show substantial (biologically significant) increases in the number of revertants versus solvent controls (Rt / RC < 2) and no experiment gave evidence of rising trend in the number of revertants with increasing dose.
Neither increasing of number of revertants nor dose-dependence was observed in experiments with S. typhimurium TA 1537 and E. coli WP2 uvrA with metabolic activation.
Increased values of numbers of revertants as well as dose-dependence were observed in three Salmonella strains with metabolic activation. These results were confirmed with repeated experiments.

COMPARISON WITH HISTORICAL CONTROL DATA:
All the control numbers were compared with historical ranges of mutant frequencies obtained in VUOS laboratory. The actual numbers were in ranges of the historical numbers.

Conclusions:

The test material was mutagenic for tester strains S. typhimurium TA 100, TA 98 and TA 1535 with metabolic activation.

The test substance was non-mutagenic for all the S. typhimurium as well as the E. coli strain without metabolic activation, and for S. typhimurium TA 1537 and E. coli WP2 uvrA with metabolic activation.

Executive summary:

Test material was assessed for mutagenicity according to EU method B.13/14 Mutagenicity – Reverse mutation test using bacteria, which is analogous to the OECD Test Guideline No. 471.

Four indicator Salmonella typhimurium strains, TA 98, TA 100, TA 1535, TA 1537 and one indicator Escherichia coli WP2 uvrA strain, were used. The test material was dissolved in DMSO and assayed in doses of 15-1500 µg, which were applied to plates in volume of 0.1 mL.

Experiments were performed without as well as with metabolic activation with a supernatant of rat liver and a mixture of cofactors.

The test substance was mutagenic for tester strains S. typhimurium TA 100, TA 98 and TA 1535 with metabolic activation.

The test substance was non-mutagenic for all the used tester strains without metabolic activation as well as for S. typhimurium TA 1537 and E. coli WP2 uvrA with metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6.9.2012 - 08.5.2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: The cell line was obtained from ATCC
- Suitability of cells: Chinese hamster ovary (CHO-K1) cells which show a low, stable background frequency of micronucleus formation were used. CHO-K1 cells grow as an adherent monolayer in vitro. They are originally derived from the ovary of a Chinese hamster and have frequently been used in this type of test system.
- Methods for maintenance in cell culture if applicable: The concentration of the cytokinesis inhibitor was optimised for the CHO cell type and should produce a good yield of 5E+05 binucleate cells/10 mL for scoring.
- Modal number of chromosomes: 20

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The cell line was stored in polypropylene ampoules at –196 °C in Ham’s F-12K1 medium and 10 % dimethylsulfoxide until cultivation. The cells were routinely grown and sub-cultured in Ham’s F-12K medium supplemented with 10 % foetal calf serum2 and 1 % Penicillin/Streptomycin at +37 °C in a humid atmosphere containing 5 % carbon dioxide in plastic tissue culture plate
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes, once per month
- Periodically checked for karyotype stability: Yes, once per month
- Periodically 'cleansed' against high spontaneous background: Yes, once per month

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiments without metabolic activation:
15.63, 31.3, 62.5, 125 µg/mL (1.experiment), 7.81, 15.63, 31.3, 62.5, 125 µg/mL (2.experiment)

Experiments with metabolic activation:
7.81, 15.63, 31.3, 62.5, 125 µg/mL

The highest concentration should aim to produce 55 ± 5% cytotoxicity. Higher levels may induce chromosome damage as a secondary effect of cytotoxicity. Where cytotoxicity occurs, the test concentrations selected should cover a range from that producing 55 ± 5% cytotoxicity, to little or no cytotoxicity. Cytotoxicity (cytostasis) is quantified from the cytokinesis proliferation block index (CPBI).
If no cytotoxicity or precipitate is observed, the highest test concentration corresponds to 0.01 M, 5 mg/mL or 5 μL/mL, whichever is the lowest. The concentrations selected for analysis are, in general, separated by a spacing of no more than √10. If the test item exhibit a steep concentration-response curve, it is necessary to more closely space the test item concentrations so that cultures in the moderate and low toxicity ranges also are scored.
When solubility is a limiting factor, the maximum concentration would be the lowest at which minimal precipitate is visible in cultures, if there is no interference with scoring. Evaluation of precipitation is done by methods such as light microscopy, precipitate would be evaluated at the beginning and end of the treatment period.
At least three analysable test concentrations were evaluated in the main study. In order to select the suitable concentrations, one preliminary cytotoxicity test without (20 h exposure) and with metabolic activation (4 h exposure) was performed. In this preliminary experiment without and with metabolic activation test item precipitation was noted in the experiments without and with metabolic activation starting at concentrations of 125 μg test material/mL. Hence, 125 μg test material/mL were employed as the top concentration for the genotoxicity tests without and with metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Remarks:

untreated culture

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: colchicine

Remarks:

Without metabolic activation: mitomycin C and colchicine, with metabolic activation: cyclophoshamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 20 hrs - without metabolic activation, 4 hrs with metabolic activation.
- Fixation time (start of exposure up to fixation or harvest of cells): The harvesting time was 20 hours after the end of exposure.

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B at 5 µg/mL.

STAIN (for cytogenetic assays): 10 % Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 1000 binucleated cells per duplicate cell culture were scored to assess the frequency of cells with one, two, or more than two micronuclei. Additionally, the cells were classified as mononucleates, binucleates or multinucleates to estimate the proliferation index as a measure of toxicity.

DETERMINATION OF CYTOTOXICITY
- Method: CBPI (Cytokinesis-Block Proliferation Index) or Replicative Index (RI)

- OTHER:
The test material was completely dissolved in dimethylsulfoxide (DMSO). A correction factor of 1.1 was used in order to correct for the purity of the test item of 90.0 % only. Preparations of the test item made on the day of use were employed.

- Treatment Schedule
Without S9 mix:
4-hours exposure:
Cultures were initiated and maintained as described above. After 24 hours the medium was replaced by 9.9 mL of fresh F-12K medium without FCS. Subsequently 100 μL of the negative control, 5 concentrations of the test item or 2 or one concentrations of the positive controls Mitomycin C or Colchicine were added to each culture. The cultures were then incubated for 4 hours at +37 °C and 5 % CO2. Afterwards the medium was removed and the cultures were washed two times with PBS. After addition of 10 mL F12K-medium containing 10 % FCS with 5 μg/mL Cytochalasin B the cultures were incubated for further 20 hours at 37°C and 5 % CO2.
20-hours exposure:
Cultures were initiated and maintained as described above. After 24 h the medium was replaced by 9.9 mL of fresh F12K-medium with FCS and 100 μL of the negative control, 5 concentrations of the test item or 2 or one concentrations of the positive control (Mitomycin C or Colchicine) were added to each culture in F12K-medium containing 10% FCS. The cultures were incubated for 20 hours at 37 °C and 5 % CO2. Afterwards the medium was removed and the cultures were washed two times with PBS. After addition of 10 mL F12K-medium containing 10 % FCS with 5 μg/mL Cytochalasin B the cultures were incubated for further 20 hours at 37 °C and 5 % CO2.
With S9 mix:
4-hours exposure:
Cultures were initiated and maintained as described above. After 24 h the medium was carefully removed and replaced by 9.9 mL premixed medium containing 50 % F12K-medium and 50 % S9-Mix. Afterwards 100 μL of the negative control, 5 concentrations of the test item or 2 or one concentrations of the positive controls Mitomycin C or Colchicine were added to each culture. The cultures were then incubated for 4 hours at +37 °C and 5 % CO2. Afterwards the medium was removed and the cultures were washed two times with PBS. After addition of 10 mL F12K-medium containing 10 % FCS with 5 μg/mL Cytochalasin B the cultures were incubated for further 20 hours at 37 °C and 5 % CO2.

- Culture harvesting and slide preparation
After the end of the 4- or 20-hour treatment period, mitotic activity was arrested by the addition of CytoB to each culture at a final concentration of 5 μg/mL. After a 20-hour incubation period the medium was removed and discarded. The cells were trypsinised (0.5 mL trypsin was added). The cells were removed, harvested and placed in a plastic conical centrifuge tube. The cell suspensions were then centrifuged for 5 minutes at 1250 rpm. The supernatant was discarded and the cells resuspended in 2.5 mL 1 % sodium citrate solution. After 15 minutes incubation in water bath at 37 °C, the cell suspensions were centrifugated for 5 minutes at 800 rpm. The supernatant was discarded and 5 mL of freshly prepared fixative (3 parts methanol : 1 part glacial acetic acid v/v) added. The pellets were resuspended and left at room temperature for 30 minutes and then centrifugated for 5 minutes at 800 rpm. The fixative was removed and discarded. 0.6 mL fresh fixative and 0.15 mL 30 % acetic acid were added and the cell pellet resuspended. Single drops of the cell suspension were spread on clean, grease-free slides on a hot plate (approx. 50°C) and the slides were carefully pivoted.
The slides were left to air-dry at room temperature, then stained in 10 % Giemsa.

- Analysis
The evaluation of cytotoxicity was based on the Cytokinesis-Block Proliferation Index (CBPI) or the Replicative Index (RI).
The CBPI indicates the average number of cell cycles per cell during the period of exposure to cytoB, and is used to calculate cell proliferation.
The RI indicates the relative number of nuclei in treated cultures compared to control cultures and can be used to calculate the % cytostasis:

CBPI = ((No. mononucleate cells) + (2 x No. binucleate cells) + (2 x No. multinucleate cells)) / total number of cells

Thus, a CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytostasis.

Cytostasis = 100 - RI

RI = (((No. binucleated cells) + (2 x No. multinucleate cells)) / (total number of cells)T) / ((No. binucleated cells) + (2 x No. multinucleate cells)) / (total number of cells)C)) x 100

Where
T = treated controls
C = control cultures

Thus, an RI of 53 % means that, compared to the numbers of cells that have divided to form binucleate and multinucleate cells in the control culture, only 53 % of this number divided in the treated culture, i.e. 47 % cytostasis.
All slides, including those of the solvent controls, were independently coded before the microscopic analysis.

Evaluation criteria:

If a test item induces a concentration-related increase or a statistical significant and reproducible increase in the number of cells containing micronuclei, it is classified as a positive result.

Statistics:

The assessment was carried out by a comparison of the samples with the positive and the vehicle control using a chi-square test corrected for continuity according to YATES (COLQUHOUN, 1971) as recommended by the UKEMS guidelines (The United Kingdom Branch of the European Environmental Mutagen Society: Report of the UKEMS subcommittee on guidelines for mutagenicity testing, part III, 1989: Statistical evaluation of mutagenicity data). However, the results of statistical testing were assessed with respect to dose-response relationship. Reproducibility and historical data were also be taken into consideration.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Tests without metabolic activation (4- and 20-hour exposure)
The micronucleus frequencies of cultures treated with the test material at concentrations from 15.63 or 7.81 to 125 μg/mL medium (4-h and 20-h exposure, respectively) in the absence of metabolic activation ranged from 2.5 to 24.0 micronuclei per 1000 binucleated cells. Mean incidences of micronucleus frequencies of 4.0 or 24.5 micronuclei per 1000 binucleated cells were observed after a 4-hour and 20-hour exposure to vehicle DMSO, respectively. These results are considered to be within the range of the historical controls of micronucleus frequencies of the solvent controls: 1.0 to 26.0 micronuclei per 1000 binucleated cells, most recent background data for the last 27 studies (not audited by the QAU-department).
All incidences of second experiment (controls and test item groups) were slightly higher than in the first experiment, but within the normal background data range and further, no increase was observed.

Test with metabolic activation (4-hour exposure)
The micronucleus frequencies of cultures treated with Solvent Red 19E at concentrations from 7.81 to 125 μg/mL medium in the presence of metabolic activation ranged from 16.5 to 23.0 micronuclei per 1000 binucleated cells. Mean incidences of micronucleus frequencies of 19.5 or 17.5 micronuclei per 1000 binucleated cells were observed in the first and second experiment with the vehicle DMSO, respectively.
The results obtained are considered to be within the range of untreated controls: 12.5 micronuclei per 1000 binucleated cells and the historical control range of micronucleus frequency of the solvent controls: 2 to 25 micronuclei per 1000 binucleated cells, most recent background data for the last 27 studies (not audited by the QAU-department).


TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the preliminary experiment without and with metabolic activation test item precipitation was noted in the experiments without and with metabolic activation. Hence, 125 µg Solvent Red 19E/mL were employed as the top concentration for the genotoxicity tests without and with metabolic activation. In the main study test item precipitation was noted in the experiments without and with metabolic activation at the top concentration of 125 µg Solvent Red 19E/mL.

RANGE-FINDING/SCREENING STUDIES: The concentrations employed were chosen based on the results of a cytotoxicity study.

COMPARISON WITH HISTORICAL CONTROL DATA: These results are considered to be within the range of the historical controls of micronucleus frequencies of the solvent controls, most recent background data for the last 27 studies.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test concentrations in the cytotoxicity test were 2.5, 10, 25, 100, 250 and 500 µg/mL. The precipitation of the test substance was noted at concentrations 100, 250 and 500 µg/mL.

Conclusions:

The test material revealed no indications of genotoxic properties.

Executive summary:

The test material was assessed for genotoxicity in an in vitro micronucleus test according to OECD Test Guideline 487.

The test was carried out employing 2 exposure times without S9 mix: 4 and 20 hours, and 1 exposure time with S9 mix: 4 hours. The experiment with S9 mix was carried out twice. The harvesting time was 20 hours after the end of exposure. The study was conducted in duplicate.

The test material was completely dissolved in dimethylsulfoxide (DMSO). The vehicle served as the negative control. A correction factor of 1.1 was used in order to correct for the purity of the test item of 90.0 % only.

The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation test item precipitation was noted starting at concentrations of 125 µg test material/mL in the experiments without and with metabolic activation. Hence, 125 µg test material/mL were employed as the top concentration for the genotoxicity tests without and with metabolic activation.

In the main study test item precipitation was noted in the experiments without and with metabolic activation at the top concentration of 125 µg test material/mL.

Mitomycin C and colchicine were employed as positive controls in the absence and cyclophosphamide was used in the presence of metabolic activation.

 

Tests without metabolic activation (4- and 20-hour exposure)

The micronucleus frequencies of cultures treated at concentrations from 15.63 or 7.81 to 125 µg/mL medium (4 h and 20-h exposure, respectively) in the absence of metabolic activation ranged from 2.5 to 24.0 micronuclei per 1000 binucleated cells. Mean incidences of micronucleus frequencies of 4.0 or 24.5 micronuclei per 1000 binucleated cells were observed after a 4-hour and 20-hour exposure to vehicle DMSO, respectively. These results are considered to be within the range of the historical controls of micronucleus frequencies of the solvent controls: 1.0 to 26.0 micronuclei per 1000 binucleated cells, most recent background data for the last 27 studies.

All incidences of second experiment (controls and test item groups) were slightly higher than in the first experiment, but within the normal background data range and further, no increase was observed.

 

Test with metabolic activation (4-hour exposure)

The micronucleus frequencies of cultures treated at concentrations from 7.81 to 125 µg/mL medium in the presence of metabolic activation ranged from 16.5 to 23.0 micronucleus per 1000 binucleated cells. Mean incidences of micronucleus frequencies of 19.5 or 17.5 micronuclei per 1000 binucleated cells were observed in the first and second experiment with the vehicle DMSO, respectively.

The results obtained are considered to be within the range of untreated controls: 12.5 micronuclei per 1000 binucleated cells and the historical control range of micronucleus frequency of the solvent controls: 2 to 25 micronuclei per 1000 binucleated cells, most recent background data for the last 27 studies.

Reference 3

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

(see Overall remarks)

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

Target gene:

thymidine kinase gene (TK+/-)

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.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

3-MC

Positive control substance:

3-methylcholanthrene

Remarks:

with metabolic activation

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.

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'.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The material was examined in three guideline and GLP compliant in vitro studies covering all genotoxicity endpoints (Ames test, chromosomal aberration assay, mammalian cell mutation assay).

Based on the results of these studies and according to the mutagenicity testing strategy (Guidance on information requirements and chemical safety assessment, Chapter R.7a: Endpoint specific guidance) there is no need to perform further testing. The substance is considered as not genotoxic.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

IN VITRO

BACTERIAL REVERSE MUTATION TEST

Test material was assessed for mutagenicity according to EU method B.13/14 Mutagenicity – Reverse mutation test using bacteria, which is analogous to the OECD Test Guideline No. 471.

Four indicator Salmonella typhimurium strains, TA 98, TA 100, TA 1535, TA 1537 and one indicator Escherichia coli WP2 uvrA strain, were used. The test material was dissolved in DMSO and assayed in doses of 15-1500 µg, which were applied to plates in volume of 0.1 mL.

Experiments were performed without as well as with metabolic activation with a supernatant of rat liver and a mixture of cofactors.

The test substance was mutagenic for tester strains S. typhimurium TA 100, TA 98 and TA 1535 with metabolic activation.

The test substance was non-mutagenic for all the used tester strains without metabolic activation as well as for S. typhimurium TA 1537 and E. coli WP2 uvrA with metabolic activation.

MICRONUCLEUS TEST

The test material was assessed for genotoxicity in an in vitro micronucleus test according to OECD Test Guideline 487.

The test was carried out employing 2 exposure times without S9 mix: 4 and 20 hours, and 1 exposure time with S9 mix: 4 hours. The experiment with S9 mix was carried out twice. The harvesting time was 20 hours after the end of exposure. The study was conducted in duplicate.

The test material was completely dissolved in dimethylsulfoxide (DMSO). The vehicle served as the negative control. A correction factor of 1.1 was used in order to correct for the purity of the test item of 90.0 % only.

The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation test item precipitation was noted starting at concentrations of 125 µg test material/mL in the experiments without and with metabolic activation. Hence, 125 µg test material/mL were employed as the top concentration for the genotoxicity tests without and with metabolic activation.

In the main study test item precipitation was noted in the experiments without and with metabolic activation at the top concentration of 125 µg test material/mL.

Mitomycin C and colchicine were employed as positive controls in the absence and cyclophosphamide was used in the presence of metabolic activation. 

- Tests without metabolic activation (4- and 20-hour exposure)

The micronucleus frequencies of cultures treated at concentrations from 15.63 or 7.81 to 125 µg/mL medium (4 h and 20-h exposure, respectively) in the absence of metabolic activation ranged from 2.5 to 24.0 micronuclei per 1000 binucleated cells. Mean incidences of micronucleus frequencies of 4.0 or 24.5 micronuclei per 1000 binucleated cells were observed after a 4-hour and 20-hour exposure to vehicle DMSO, respectively. These results are considered to be within the range of the historical controls of micronucleus frequencies of the solvent controls: 1.0 to 26.0 micronuclei per 1000 binucleated cells, most recent background data for the last 27 studies.

All incidences of second experiment (controls and test item groups) were slightly higher than in the first experiment, but within the normal background data range and further, no increase was observed.

- Test with metabolic activation (4-hour exposure)

The micronucleus frequencies of cultures treated at concentrations from 7.81 to 125 µg/mL medium in the presence of metabolic activation ranged from 16.5 to 23.0 micronucleus per 1000 binucleated cells. Mean incidences of micronucleus frequencies of 19.5 or 17.5 micronuclei per 1000 binucleated cells were observed in the first and second experiment with the vehicle DMSO, respectively.

The results obtained are considered to be within the range of untreated controls: 12.5 micronuclei per 1000 binucleated cells and the historical control range of micronucleus frequency of the solvent controls: 2 to 25 micronuclei per 1000 binucleated cells, most recent background data for the last 27 studies.

MOUSE LYMPHOMA ASSAY

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.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.