trisodium 3-[(E)-2-(4-{[4-chloro-6-({2-[(4-{[4-(ethenesulfonyl)phenyl]amino}-6-fluoro-1,3,5-triazin-2-yl)amino]propyl}amino)-1,3,5-triazin-2-yl]amino}-5-sulfonaphthalen-1-yl)diazen-1-yl]naphthalene-1,5-disulfonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reactive Yellow 214 was mutagenic in the bacterial reverse mutation assay and clastogenic in the in vitro chromosomal aberration test. However in higher tier studies such as in vitro HPRT and in vivo micronucleus test, it did not reproduce the mutagenic or clastogenic effect. Basis of these two higher tier studies, the substance is considered as non genotoxic.

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:

10 May 2006 to 31 July 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: Japanese Guideline: Kanpoan No. 287 - Environment Protection Agency

Qualifier:

according to guideline

Guideline:

other: Japanese Guideline: Eisei No. 127 - Ministry of Health & Welfare

Qualifier:

according to guideline

Guideline:

other: Japanese Guideline: Heisei 09/10/31 Kikyoku No. 2 - Ministry of International Trade & Industry

GLP compliance:

yes (incl. QA statement)

Remarks:

RCC Cytotest Cell Research GmbH, In den Leppsteinwiesen 19, 64380 Roβdorf

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Identity: FAT 40826/A
Batch: TZ 5604 BOP 01/06
Purity: Content of organic part (Na-salt): approx. 78 %; Oligomers: 13 %; Main component: approx. 48 %
Appearance: Solid, orange powder
Stability in solvent: Stable for 7 days in water, saline, polyethylene glycol, and CMC at room temperature
Expiration date: February 01, 2011
Storage: At room temperature at about 20 °C in a desiccator.

Target gene:

histidine and tryptophan

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate
Experiment II: 33, 100, 333, 1000, 2500, and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2-AA

Remarks:

Strain TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA, with metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Strain WP2 uvrA, without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Strain TA 1535 and TA 100, without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

Strain TA 1537 and TA 98, without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar

NUMBER OF REPLICATIONS: 3

STORAGE AND PRECULTURE
The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen. From the thawed ampoules of the strains 0.5 mL suspension was transferred into 250 mL Erlenmeyer flasks containing 20 mL nutrient medium. A solution of 20 µL ampicillin (25 µg/mL) was added to the strains TA 98 and TA 100. The bacterial cultures were incubated in a shaking water bath for 4 hours at 37 °C.

DETERMINATION OF CYTOTOXICITY
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as for the experiment I (plate incorporation test). Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn. The pre-experiment is reported as main experiment I, if the following criteria are met: Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

DOSE SELECTION
In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as experiment I since no relevant toxic effects were observed and 5000 µg/plate were chosen as maximal concentration.

EXPERIMENTAL PERFORMANCE
In the pre-incubation assay 100 µL test solution, 500 µL S9 mix / S9 mix substitution buffer, 100 µL bacterial suspension were mixed in a test tube and shaken at 37 °C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on selective agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark. The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB 7BN, UK) with the software program Ames Study Manager.

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY:
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria: regular background growth in the negative and solvent control, the spontaneous reversion rates in the negative and solvent control are in the range of our historical data, the positive control substances should produce a significant increase in mutant colony frequencies.

EVALUATION OF RESULTS
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

No statistical evaluation of the data is required.

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Conclusions:

FAT 40826/A is considered to be mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

In a GLP-compliant reverse mutation assay, performed according to OECD guideline 471, 4 Salmonella typhimurium strains (TA 1535, TA 1537, TA 98, and TA 100) and 1 Escherichia coli strain WP2 uvrA, were used to the test the mutagenic potential of the test substance (33, 100, 333, 1000, 2500, 5000 µg per plate), both with and without metabolic activation. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. No toxic effects occurred in the test groups with and without metabolic activation. A moderate but dose dependent increase in revertant colony numbers was observed following treatment with the test substance in strains TA 98 (without metabolic activation) and TA 100 with and without metabolic activation in both experiments. In experiment I, the required threshold of two times the number of the corresponding solvent control could not be reached. However, the number of colonies was above the laboratory's historical control range in strains TA 98 and TA 100 from 1000 up to 5000 µg/plate without metabolic activation and in strain TA 100 at 2500 and 5000 µg/plate with metabolic activation. Based on these equivocal results, the more sensitive pre-incubation design was chosen for experiment II. Again, a moderate but dose dependent increase in revertant colony numbers was observed in strain TA 98 without S9 mix and in strain TA 100 with S9 mix. The threshold was exceeded in strain TA 98 at 5000 µg/plate without S9 mix (factor 2.3). In strain TA 100, the required threshold was not quite reached but a dose dependent response was observed in the presence of metabolic activation. Therefore, a possible mutagenic potential of the test item cannot be excluded. In conclusion, FAT 40826/A induced gene mutations by base pair changes or frameshifts in the genome of the strains TA 98 and TA 100.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 May 2006 to 19 September 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

"Kanpoan No. 287 - Environmental Agency"
"Eisei No. 127 - Ministry of Health & Welfare"
"Heisei 09/10/31 Kikyoku No. 2 - Ministry of International Trade & Industry".

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Identity: FAT 40826/A
Batch no.: TZ 5604 BOP 01/06
Expiration date: February 01, 2011
Purity: Content of organic part (Na-salt): approx. 78 %; Oligomers: 13 %; Main component: approx. 48 %
Solubility in water: Approx. >50 g/L at room temperature
Stability in water: Max. 7 days at room temperature
pH: 7.6 (1 g/L)
Aggregate state/physical form at room temperature: Solid (orange powder)
Storage conditions: At room temperature at about 20 °C, away from direct sunlight
Specific instructions: Store in desiccator

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

- Without S9 mix: 40, 80, 160, 320, 640, 1280 µg/mL
- With S9 mix: 10, 20, 40, 80, 160, 320 µg/mL

Vehicle / solvent:

Deionised water. The final concentration of deionised water in the culture medium was 10 % (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

RANGE-FINDER
A pre-test on cell growth inhibition with 4 hours and 24 hours treatment was performed in order to determine the toxicity of the test item. Cytotoxicity was determined using concentrations separated by no more than a factor of 2 - square root 10. The general experimental conditions in this pre-test were the same as described below for the cytogenetic main experiment. The following method was used: In a quantitative assessment, exponentially growing cell cultures (seeding about 40,000 cells/slide, with regard to the culture time 48 hours) were treated with the test item for simulating the conditions of the main experiment. A qualitative evaluation of cell number and cell morphology was made 4 hours and 24 hours after start of treatment. The cells were stained 24 hours after start of treatment. Using a 400 fold microscopic magnification the cells were counted in 10 coordinate defined fields of the slides (2 slides per treatment group).

EXPERIMENTAL PERFORMANCE
- Exponentially growing stock cultures more than 50 % confluent are treated with trypsin-EDTA-solution at 37°C for approx. 5 minutes. Then the enzymatic treatment is stopped by adding complete culture medium and a single cell suspension is prepared. The trypsin concentration for all subculturing steps is 0.5 % (w/v) in Ca-Mg-free salt solution (Invitrogen GIBCO, D-76131 Karlsruhe). Prior to the trypsin treatment the cells are rinsed with Ca-Mg-free salt solution. The cells were seeded into Quadriperm dishes (Heraeus, D-63450 Hanau) which contained microscopic slides (at least 2 chambers per dish and test group). In each chamber 1E4 - 6E4 cells were seeded with regard to the preparation time. The medium was MEM with 10 % FCS (complete medium).
- Exposure duration: The culture medium of exponentially growing cell cultures was replaced with serum-free medium (for treatment with S9 mix) or complete medium (for treatment without S9 mix) with 10 % FCS (v/v), containing the test item. For the treatment with metabolic activation 50 µL S9 mix per mL medium were used. Concurrent solvent and positive controls were performed. After 4 hours the cultures were washed twice with "Saline G" and then the cells were cultured in complete medium for the remaining culture time.
- Preparation of the cultures: Colcemid was added (0.2 µg/mL culture medium) to the cultures 15.5 hours after the start of the treatment. The cells on the slides were treated 2.5 hours later in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37° C. After incubation in the hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3:1 parts, respectively). Per experiment two slides per group were prepared. After preparation the cells were stained with Giemsa (E. Merck, D-64293 Darmstadt).
- Evaluation of Cell Numbers: For evaluation of cytotoxicity indicated by reduced cell numbers additional two cultures per test item and solvent control group, not treated with colcemid, were set up in parallel. These cultures were stained after 18 hours, in order to determine microscopically the cell number within 10 defined fields per coded slide. The cell number of the treatment groups is given in percentage compared to the respective solvent control.
- Analysis of Metaphase Cells: Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetic) using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells in 500 metaphase plates per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).

Evaluation criteria:

- Acceptability of the test: The chromosome aberration test performed in our laboratory is considered acceptable if it meets the following criteria:
a) The number of structural aberrations found in the solvent controls falls within the range of our historical laboratory control data: 0.0 - 4.0 %.
b) The positive control substances should produce significant increases in the number of cells with structural, chromosome aberrations, which are within the range of the laboratory's historical control data.
- Evaluation of results: A test item is classified as non-clastogenic if: the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps) and/or no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as clastogenic if: the number of induced structural chromosome aberrations is not in the range of the historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps) and either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.
A test item can be classified as aneugenic if: the number of induced numerical aberrations is not in the range of our historical control data (0.0 - 8.5 % polyploid cells).

Statistics:

Statistical significance was confirmed by means of the Fisher's exact test (p <0.05). However, both biological and statistical significance should be considered together.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE FINDING STUDY:
In a range finding pre-test on toxicity cell numbers were scored 24 hrs after start of treatment as an indicator for cytotoxicity. Concentrations between 39.1 and 5000 µg/mL were applied. Clear toxic effects were observed after treatment with 156.3 µg/mL and above in the absence and presence of S9 mix. In addition, 24 hours continuous treatment with 78.1 µg/mL and above in the absence of S9 mix induced strong toxic effects.

TEST-SPECIFIC CONFOUNDING FACTORS:
In the pre-experiment, neither precipitation of the test item in culture medium nor a relevant influence of the test item on the pH value or osmolarity was observed (solvent control 265 mOsm, pH 7.3 versus 308 mOsm and pH 7.4 at 5000 µg/mL).

MAIN TEST:
In this study, dose-related increases in the number of aberrant cells were observed at 80 to 320 µg/mL in the absence of S9 mix (3.0 %, 11.5 %, and 12.0 % aberrant cells exclusive gaps, respectively) and at 40 to 160 µg/mL in the presence of S9 mix (2.0 %, 5.0 %, and 11.0 % aberrant cells exclusive gaps, respectively). The increase in the number of aberrant cells, exclusive gaps at 160 µg/mL and 320 µg/mL (11.5 % and 12.0 %, respectively) in the absence of S9 mix and at 160 µg/mL (11.0 %) in the presence of S9 mix, were statistically significant when compared to the corresponding solvent controls (1.0 % and 2.5 %, respectively). The values at the two highest evaluated concentrations, in the absence and presence of S9 mix, clearly exceeded our historical control data range (0.0 - 4.0 % aberrant cells exclusive gaps). Therefore, the statistical significances and the dose-dependencies have to be regarded as being biologically relevant. No biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (1.4 - 3.4 %) as compared to the rates of the solvent controls (2.3 - 3.3 %).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the main experiment, in the absence of S9 mix, toxic effects indicated by clearly reduced cell numbers of below 50 % of control were observed after 4 hours treatment with 320 µg/mL (26.0 % of control). In the presence of S9 mix, concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage.

Remarks on result:

other: all strains/cell types tested

Conclusions:

FAT 40826/A induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) in the prensence or absence of S9 mix.

Executive summary:

In a GLP-compliant chromosome aberration test, tested according to OECD guideline 473, Chinese hamster V79 cells, were exposed to the test substance, with and without metabolic activation by S9 mix. The exposure period was 4 hours with and without metabolic activation. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations. Dose selection of the main experiments was based on a pretest. In the absence of S9 mix, clear cytotoxicity was observed at the highest evaluated concentration. In the presence of S9 mix, concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage. In the absence and the presence of S9 mix, dose-related significant increases in the number of cells carrying structural chromosomal aberrations were observed after treatment with the test substance. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. In conclusion, it can be stated that under the experimental conditions reported, the test substance induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, the test substance is considered to be clastogenic in this chromosome aberration test in the absence and presence of S9 mix.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

HPRT

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 July 2006 to 30 October 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

"Kanpoan No. 287 - Environment Protection Agency"
"Eisei No. 127 - Ministry of Health & Welfare"
"Heisei 09/10/31 Kikyoku No. 2 - Ministry of International Trade & Industry"

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

Identity: FAT 40826/A
Batch no.: TZ 5604 BOP 01/06
Expiration date: February 01, 2011
Purity: Content of organic part (Na-salt): approx. 78 %; Oligomers: 13 %; Main component: approx. 48 %
Solubility in water: Approx. >50 g/L at room temperature
Stability in water: Max. 7 days at room temperature
pH: 7.6 (1 g/L)
Aggregate state/physical form at room temperature: Solid (orange powder)
Storage conditions: At room temperature at about 20 °C, away from direct sunlight
Specific instructions: Store in desiccator

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Prior to mutagenicity testing the amount of spontaneous mutants is reduced by growing the cells for one day in HAT medium.
- Each batch is screened for mycoplasma contamination and checked for karyotype stability and spontaneous mutant frequency.

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment I
without S9 mix: 1.25, 2.5, 5, 10, 20, 30, 40 µg/mL
with S9 mix: 12.5, 25, 50, 100, 150, 200 µg/mL

Experiment II
without S9 mix: 9.4, 18.8, 37.5, 75, 150, 225, and 300 µg/mL

Vehicle / solvent:

water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

DETERMINATION OF CYTOTOXICITY
A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. The general culture conditions and experimental conditions in this pre-test were the same as described for the mutagenicity experiment below. In this pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE).

MAIN EXPERIMENT
Seeding: Three days old exponentially growing stock cultures (more than 50 % confluent) were trypsinized at 37 °C for 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium and a single cell suspension was prepared. The trypsin concentration for all subculturing steps was 0.2 % in Ca-Mg-free salt solution (Trypsin: Difco Laboratories, Detroit, USA). Prior to the trypsin treatment the cells were rinsed with Ca-Mg-free salt solution containing 200 mg/L EDTA (ethylene diamine tetraacetic acid). The cell suspension was seeded into plastic culture flasks (Greiner, D-72632 Frickenhausen). Approximately 1.5E6 (single culture) and 5E2 cells (in duplicate) were seeded in MEM with 10 % FCS (complete medium) for the determination of mutation rate and toxicity, respectively.
Treatment: After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 µL/mL S9 mix. Concurrent negative and positive controls were treated in parallel. After 4 hours this medium was replaced with complete medium following two washing steps with "saline G". In the second experiment the cells were exposed to the test item for 24 hours in complete medium in the absence of metabolic activation. The pH was adjusted to 7.2. The colonies used to determine the cloning efficiency (survival) were fixed and stained approx. 7 days after treatment as described below. Three days after treatment 1.5xE6 cells per experimental point were subcultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days, five 80 cm² cell culture flasks were seeded with about 3 - 5E5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in nonselective medium to determine the viability. The cultures are incubated at 37 °C in a humidified atmosphere with 4.5 % C02 for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution (E. MERCK, D-64293 Darmstadt). The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope (Nikon, D-40407 Düsseldorf). Subculturing of a log-phase culture showed an initial spontaneous mutation rate at the beginning of the experiment of 9.6 mutant colonies (Experiment I) and 4.9 (Experiment II) mutant colonies per 1E6 cells.

Evaluation criteria:

- Acceptability of the assay:
The gene mutation assay is considered acceptable if it meets the following criteria: the numbers of mutant colonies per 1E6 cells found in the negative and/or solvent controls fall within the laboratory historical control data range of 2000-2005, the positive control substances must produce a significant increase in mutant colony frequencies, the cloning efficiency II (absolute value) of the negative and/or solvent controls must exceed 50 %.
- Evaluation of results:
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 frequency at least at one of the concentrations 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 negative control data. If there is by chance a low spontaneous mutation rate in the range normally found (0.5 - 31.8 mutants per 1E6 cells) a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of negative and solvent controls within all experiments of this study was also taken into consideration.

Statistics:

A linear regression was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT® statistics software. The number of mutant colonies obtained in the groups treated with the test item was 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 and statistical significance should be considered together.

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

Positive controls validity:

valid

Additional information on results:

RESULTS GENOTOXICITY:
No relevant and reproducible increase of the mutation frequency was observed in the main experiments up to the maximum concentration. All mutation frequencies remained well within the historical data range of negative and solvent controls. The threshold of three times the mutation frequency of the corresponding solvent control was exceeded in the first culture of the second experiment at the lowest and the highest analysed concentration (18.8 and 225 μg/mL). However, this increase was not dose dependent and not reproduced in the parallel culture under identical conditions. The absolute values of the mutation frequency remained well within the historical control range. Therefore, this increase was judged as biologically irrelevant. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT statistics software. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was calculated in the first experiment in culture II with and without metabolic activation. However, the absolute values of the individual mutation frequencies remained low (max. 14.3 mutant colonies per 1E6 cells) and did not exceed the historical control range by far. Therefore, the trends described were judged as biologically irrelevant.

TEST-SPECIFIC CONFOUNDING FACTORS:
- No precipitation was observed in any of the main experiments.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Relevant toxic effects as indicated by a relative cloning efficiency I of 50 % or less were noted in the first experiment at 30 and 40 μg/mL (culture I only) and 200 μg/mL with metabolic activation. In the second experiment toxic effects as described above were observed at 225 μg/mL.

Remarks on result:

other: all strains/cell types tested

Conclusions:

The test substance is considered to be non-mutagenic in this HPRT assay.

Executive summary:

In a GLP-compliant mammalian cell gene mutation test, performed according to OECD guideline 476, V79 cells of the Chinese hamster were exposed to the test substance with and without metabolic activation to investigate the potential of the test substance to induce gene mutations at the HPRT locus. The assay was performed in two independent experiments. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 h. The highest concentration (5000 ug/mL) used in the range finding pre-experiment was chosen with respect to the current OECD guideline 476. The dose range of the main experiments was limited by toxicity of the test item. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. In conclusion, FAT 40826/A did not induce gene mutations at the HPRT locus in V79 cells and therefore is considered to be non-mutagenic in the HPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Reactive Yellow 214 is considered to be non-mutagenic in the micronucleus assay.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 October 2006 to 02 March 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Specific details on test material used for the study:

Identity: FAT 40826/A
Batch no.: TZ 5604 BOP 01/06
Expiration date: February 01, 2011
Purity: Content of organic part (Na-salt): approx. 78 %; Oligomers: 13 %; Main component: approx. 48 %
Solubility in water: Approx. >50 g/L at room temperature
Stability in water: Max. 7 days at room temperature
pH: 7.6 (1 g/L)
Aggregate state/physical form at room temperature: Solid (orange powder)
Storage conditions: At room temperature at about 20 °C, away from direct sunlight
Specific instructions: Store in desiccator

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The mouse is an animal that has been used for many years as a suitable experimental animal in cytogenetic investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies. Data from such experiments also may be useful for the design and the performance of the micronucleus test.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, D-33178, Borchen
- Age at start acclimatisation: 7-8 weeks
- Weight at start of treatment: Males 31.5 g (SD ± 2.1 g), Females: 31.1g (SD ± 1.6 g)
- Assigned to test groups randomly: yes
- Housing: Individually, in Makrolon Type I cages, with wire mesh top (EHRET GmbH, D-79302 Emmendingen) with granulated soft wood bedding (Harlan Winkelmann GmbH, D-33178 Borchen).
- Diet: Pelleted standard diet, ad libitum (Harlan Winkelmann GmbH, D-33178 Borchen).
- Water: tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Frequency of treatment:

Single treatment

Post exposure period:

24 hours for all doses, 48 hours for the 2000 mg/kg bw dose group.

Remarks:

Doses / Concentrations:
500, 1000, 2000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide;
- Route of administration: orally
- Doses: 40 mg/kg bw
- Volume administrated: 10 mL/kg bw

Tissues and cell types examined:

Normochromatic and polychromatic erythrocytes

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
- The animals were sacrificed using CO2 following by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (MERCK, D-64293 Darmstadt)/Giemsa (MERCK, D-64293 Darmstadt). Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
- Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 2000 PCEs. The analysis was performed with coded slides. Ten animals (5 males, 5 females) per test group were evaluated as described.

Evaluation criteria:

Acceptance Criteria: the study was considered valid as the following criteria are met:
- the negative controls are in the range of our historical control data
- the positive controls are in the range of our historical control data
- at least 4 animals per group and sex can be evaluated
- PCE to erythrocyte ratio should not be less than 20 % of the negative control.

Evaluation of results: a test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

Statistical methods (nonparametric Mann-Whitney test) will be used as an aid in evaluating the results. However, the primary point of consideratrion is the biological relevance of the results.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

in vivo

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- The mean number of polychromatic erythrocytes was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that FAT 40826/A did not have any cytotoxic properties in the bone marrow. However, the urine of the animals treated with the mid and high dose had taken the colour of the test item, indicating the systemic distribution of the test item and thus its bioavailability.
- In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with FAT 40826/A were below or near to the value of the vehicle control group.

In the first pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 100 mg/kg b.w. FAT 40826/A formulated in deionised water. The volume administered was 5 mL/kg b.w.. In the second pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 1000 mg/kg b.w. FAT 40826/A formulated in deionised water. The volume administered was 10 mL/kg b.w.. The animals treated with 100 and 1000 mg/kg b.w. did not express any toxic reactions. In the third pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg b.w. FAT 40826/A formulated in deionised water. The volume administered was 10 mL/kg b.w.. The animals treated with 2000 mg/kg b.w. expressed toxic reactions.

 

Toxic Symptoms in the Main Experiment

The animals treated with 2000 mg/kg b.w. expressed toxic reactions such as reduction of spontaneous activity, ruffled fur and urine colour. The animals treated with 1000 mg/kg b.w. showed urine colour, However, the animals treated with 500 mg/kg and the vehicle control (deionised water) did not express any toxic reactions.

Conclusions:

FAT 40826/A did not induce micronuclei in bone marrow cells of the mouse.

Executive summary:

In a GLP-compliant erythrocyte micronucleus test, carried out according to OECD guideline 474, six NMRI mice per sex were treated once by oral gavage with the test substance (500, 1000, 2000 mg/kg bw) dissolved in water followed by a 24 or 48 hours post exposure period. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei and at least 2000 polychromatic erythrocytes (PCEs) per animal were scored. After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that the test substance did not exert any cytotoxic effects in the bone marrow. However, the urine of the animals treated with the mid and high dose had taken the colour of the test item, indicating the systemic distribution of the test item and thus its bioavailability. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. Additionally, the animals treated with 2000 mg/kg b.w. expressed toxic reactions such as reduction of spontaneous activity, ruffled fur and urine colour. The animals treated with 1000 mg/kg b.w. showed urine colour, However, the animals treated with 500 mg/kg and the vehicle control (deionised water) did not express any toxic reactions. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test substance is considered to be non-mutagenic in this micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro: Reverse mutation assay:

In a GLP-compliant reverse mutation assay, performed according to OECD guideline 471, 4 Salmonella typhimurium strains (TA 1535, TA 1537, TA 98, and TA 100) and 1 Escherichia coli strain WP2 uvrA, were used to the test the mutagenic potential of the test substance (33, 100, 333, 1000, 2500, 5000 µg per plate), both with and without metabolic activation. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. No toxic effects occurred in the test groups with and without metabolic activation. A moderate but dose dependent increase in revertant colony numbers was observed following treatment with the test substance in strains TA 98 (without metabolic activation) and TA 100 with and without metabolic activation in both experiments. In experiment I, the required threshold of two times the number of the corresponding solvent control could not be reached. However, the number of colonies was above the laboratory's historical control range in strains TA 98 and TA 100 from 1000 up to 5000 µg/plate without metabolic activation and in strain TA 100 at 2500 and 5000µg/plate with metabolic activation. Based on these equivocal results, the more sensitive pre-incubation design was chosen for experiment II. Again, a moderate but dose dependent increase in revertant colony numbers was observed in strain TA 98 without S9 mix and in strain TA 100 with S9 mix. The threshold was exceeded in strain TA 98 at 5000µg/plate without S9 mix (factor 2.3). In strain TA 100, the required threshold was not quite reached but a dose dependent response was observed in the presence of metabolic activation. Therefore, a possible mutagenic potential of the test item cannot be excluded. In conclusion, FAT 40826/A induced gene mutations by base pair changes or frameshifts in the genome of the strains TA 98 and TA 100.

 

In vitro: Chromosome aberration test:

In a GLP-compliant chromosome aberration test, tested according to OECD guideline 473, Chinese hamster V79 cells, were exposed to the test substance, with and without metabolic activation by S9 mix. The exposure period was 4 hours with and without metabolic activation. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations. Dose selection of the main experiments was based on a pretest. In the absence of S9 mix, clear cytotoxicity was observed at the highest evaluated concentration. In the presence of S9 mix, concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage. In the absence and the presence of S9 mix, dose-related significant increases in the number of cells carrying structural chromosomal aberrations were observed after treatment with the test substance. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. In conclusion, it can be stated that under the experimental conditions reported, the test substance induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, the test substance is considered to be clastogenic in this chromosome aberration test in the absence and presence of S9 mix.

 

In vitro: Gene mutation assay: HPRT

In a GLP-compliant mammalian cell gene mutation test, performed according to OECD guideline 476, V79 cells of the Chinese hamster were exposed to the test substance with and without metabolic activation to investigate the potential of the test substance to induce gene mutations at the HPRT locus. The assay was performed in two independent experiments. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 h. The highest concentration (5000 µg/mL) used in the range finding pre-experiment was chosen with respect to the current OECD guideline 476. The dose range of the main experiments was limited by toxicity of the test item. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments.

In conclusion, FAT 40826/A did not induce gene mutations at the HPRT locus in V79 cells and therefore is considered to be non-mutagenic in the HPRT assay.

 

In vivo: Micronucleus test:

In a GLP-compliant erythrocyte micronucleus test, carried out according to OECD guideline 474, six NMRI mice per sex were treated once by oral gavage with the test substance (500, 1000, 2000 mg/kg bw) dissolved in water followed by a 24 or 48 hours post exposure period. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei and at least 2000 polychromatic erythrocytes (PCEs) per animal were scored. After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that the test substance did not exert any cytotoxic effects in the bone marrow. However, the urine of the animals treated with the mid and high dose had taken the colour of the test item, indicating the systemic distribution of the test item and thus its bioavailability. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. Additionally, the animals treated with 2000 mg/kg bw expressed toxic reactions such as reduction of spontaneous activity, ruffled fur and urine colour. The animals treated with 1000 mg/kg bw showed urine colour. However, the animals treated with 500 mg/kg and the vehicle control (deionised water) did not express any toxic reactions. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test substance is considered to be non-mutagenic in this micronucleus assay.

Justification for classification or non-classification

Based on the available genotoxicity studies, Reactive Yellow 214 does not need to be classified for genotoxicity according to Regulation (EC) No. 1272/2008 (CLP).