disodium 5-{4-chloro-6-[N-ethyl-3-(vinylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-4-hydroxy-3-[(4-vinylsulfonyl)phenylazo]naphthalene-2,7-disulfonate; reaction mass of: trisodium 5-{4-chloro-6-[N-ethyl-(3-(2-sulfonatooxy)ethylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-4-hydroxy-3-[4-(vinylsulfonyl)phenylazo]naphthalene-2,7-disulfonate; tetrasodium 5-{4-chloro-6-[N-ethyl-3-(2-(sulfonatooxy)ethylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-3-[4-(2-(sulfonatooxy)ethylsulfonyl)phenylazo]-4-hydroxynaphthalene-2,7-disulfonate; trisodium 5-{4-chloro-6-[N-ethyl-3-(vinylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-4-hydroxy-3-[4-(2-(sulfonatooxy)ethylsulfonyl)phenylazo]naphthalene-2,7-disulfonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Date of Study Plan: April 11, 2002 - Date of Final Report: November 04, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted July 21, 1997

Deviations:

yes

Remarks:

concerning the acceptability of the test and more precisely Aberrant cells in % (excl. gaps). Reason for the deviation : updating. Please refer to the study report for more details.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, L1362000, Annex 4A: "Mutagenicity - In vitro Mammalian Chromosome Aberration Test"

Version / remarks:

dated May 19, 2000

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese guidelines: "Kanpoan No. 287 -- Environmental Protection Agency", "Eisei No. 127 -- Ministry of Health & Welfare", "Heisei 09110131 Kikyoku No. 2 - Ministry of lnternational rrade & lndustry".

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

444-050-5

EC Name:

-

IUPAC Name:

dodecasodium 5-({4-chloro-6-[ethyl({3-[2-(sulfooxy)ethanesulfonyl]phenyl})amino]-1,3,5-triazin-2-yl}amino)-3-[(E)-2-[4-(ethenesulfonyl)phenyl]diazen-1-yl]-4-hydroxynaphthalene-2,7-disulfonate 5-({4-chloro-6-[ethyl({3-[2-(sulfooxy)ethanesulfonyl]phenyl})amino]-1,3,5-triazin-2-yl}amino)-4-hydroxy-3-[(E)-2-{4-[2-(sulfooxy)ethanesulfonyl]phenyl}diazen-1-yl]naphthalene-2,7-disulfonate 5-[(4-chloro-6-{[3-(ethenesulfonyl)phenyl](ethyl)amino}-1,3,5-triazin-2-yl)amino]-3-[(E)-2-[4-(ethenesulfonyl)phenyl]diazen-1-yl]-4-hydroxynaphthalene-2,7-disulfonate 5-[(4-chloro-6-{[3-(ethenesulfonyl)phenyl](ethyl)amino}-1,3,5-triazin-2-yl)amino]-4-hydroxy-3-[(E)-2-{4-[2-(sulfooxy)ethanesulfonyl]phenyl}diazen-1-yl]naphthalene-2,7-disulfonate

Test material form:

solid

Details on test material:

Identity: Red Rwa 4565
Appearance: Solid, red powder
Storage: at room temperature at about 20°C

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing, LMP, Technical University Darmstadt, Ð-64287 Darmstadt)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

The highest applied concentration in the pre-test on toxicity (5310 pg/ml) was chosen respect to the current OECD Guideline 473.
Dose selection of the cytogenetic experiments was performed considering the toxicity data (for details, please refer to the study report).

Main experiment I : 175.0 µg/ml with S9 mix and 350.0 µg/ml without S9 mix
Main experiment II: 100 µg/ml (18h interval & 28h interval) without S9 mix; 262.5 µg/ml with S9 mix

Vehicle / solvent:

- Solvent used: deionised water. The final concentration of deionised water in the culture medium was 10% (v/v).
- Justification for choice of solvent: the solvent was chosen to its solubility properties and its relative nontoxicity to the cell cultures.

Controls

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

EXPERIMENTAL PERFORMANCE
SCHEDULE: please refer to section "any other information on materials and methods incl. tables" here below.

SEEDING OF THE CULTURES:
Exponentially growing stock cultures more than 50 % confluent were treated with trypsin at 37 °C for approximately 5 minutes. Then the enzymat¡c digestion was stopped by adding complete culture medium and a single cell suspension was prepared. The trypsin concentration was 0.2 % in Ca-Mg-free salt solution (Trypsin: Difco Laboratories, Detroit, USA).
The Ca-Mg-free salt solution was composed as follows (per litre): NaCl 8000 mg, KCl 200 mg, KH2PO4 200 mg and NaHPO4 150 mg.

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 cells were seeded into Quadriperm dishes (Heraeus, D-63450 Hanau) which contained microscopic slides (at least 2 chambers per dish and test group). ln each chamber 1 x 10EXP4 - 6 x 10EXP4 cells were seeded with regard to preparation time. The medium was MEM with 10 % FCS (complete medium).

TREATMENT:
Exposure period 4 hours:
The culture medium of exponentially growing cell cultures was replaced with serum-free medium (for treatment with 39 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 culture medium were added. Concurrent negative, solvent, and positive controls were performed. After 4 hrs the cultures were washed twice with "Saline G" and then the cells were cultured in complete medium for the remaining culture time.

The "saline G" solution was composed as follows (per litre): NaCl 8000 mg, KCl 400 mg, Glucose 1100 mg, Na2HPO4.7H2O 290 mg, KH2PO4 150 mg. pH was adjusted to 7.2.

Exposure period 18 and 28 hours
The culture medium of exponentially growing cell cultures was replaced with complete medium (with 10 % FCS) containing different concentrations of the test item without S9 mix. The
medium was not changed until preparation of the cells.
All cultures were incubated at 37°C in a humidified atmosphere with 4.5 % CO2 (95.5 % air).

Preparation of the Cultures:
15.5 hrs and 25.5 hrs, respectively after the start of the treatment colcemid was added (0.2 µg/ml culture medium) to the cultures. 2.5 hrs later, the cells on the slides were treated in the chambers with hypotonic solution (0.4 % KCI) for 20 min at 37°C. After incubation in the hypotonic solution the cells were fìxed with a mixture of methanol and glacial acetic acid (3 parts + 1 part). Per experiment both 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 hrs and 28 hrs, respectively, 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 lndustrie, Cytogenetik" [9]) 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. ln addition, the number of polyploid cells in 500 metaphase cells per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).

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 negative and/or 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 laboratories historical control data:

Evaluation criteria:

A test item is classifìed 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 our 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 (10) (p < 0.05).
However, both biological and statistical significance should be considered together. lf the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Ref: Richardson, C., et al (1989). Analysis of data from in vitro cytogenetic tests. Kirkland, D.J. (ed.). "Statistical evaluation of mutagenicity test data", Cambridge University Press, Cambridge, 141-154

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

Experiment II (Preparation interval of 28h)

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

Experiment II (Preparation interval of 28h)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

Experiment II and II (Preparation interval of 18h)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Strong toxic effects in exp I at 40 without and at 320 µg/ml with m. activation. ln exp. II strong toxicity at 240 µg/ml and above. Results acceptable as cell density at 1st subcultivation after treatment remained above 20% at the highest evaluated conc.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ln a range fìnding pre-test on toxicity cell numbers 24 hrs after start of treatment were scored as an indicator for cytotoxicity. Concentrations between 41.5and 5310 pg/ml were applied.
Clear toxic effects were observed after 4 hrs treatment with 663.8 µg/ml and above in the absence of S9 mix and with 331.9 µg/ml and above in the presence of S9 mix. ln addition, 24 hrs continuous treatment with 663.8 µg/ml and above in the absence of S9 mix induced strong toxic effects.

ln the pre-experiment, neither test item precipitation in culture medium 4 hrs after start of treatment nor relevant influence of the test item on the pH value or osmolarity was observed (solvent control 292 mOsm, pH 7.3 versus 305 mOsm and pH 7.4 at 5310 µg/ml.

Clearly reduced cell numbers of below 50 % were observed in experiment I in the absence of S9 mix after 4 hrs treatment at 18 hrs preparation interval with 350 µg/ml (47 % of control) and in experiment ll in the presence of S9 mix after 4 hrs treatment at 28 hrs preparation interval with 262.5 µg/ml (42% of control). No strong toxic effects indicated by reduced mitotic indices of below 50 % were observed after treatment at the evaluated test item concentrations. However, concentrations showing clearly reduced mitotic indices were not evaluable for cytogenetic damage.

ln experiment I and ll in the absence of S9 mix and in experiment I in the presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberratíon rates of the cells after treatment with the test item (0.0 - 3.0 % aberrant cells, exclusive gaps) were close to the range of the solvent control values (0.0 - 2.5 % aberrant cells, exclusive gaps) and within the range of our historical control data: 0.0 - 4.0 % aberrant cells, exclusive gaps. A single statistically significant (p < 0.05) increase was observed in the absence of S9 mix after treatment with 262.5 µg/ml . Although this increase of 3.0% aberrant cells, exclusive gaps, was statistically signifìcant compared to the low response (0.5 % aberrant cells, exclusive gaps) in the corresponding solvent control, the response is within the historical control data range (0.0 % - 4.0 % aberrant cells, exclusive gaps). Therefore, this statistical signifìcance has to be regarded as being biologically irrelevant. ln experiment ll, in the presence of S9 mix at preparation interval 28 hrs the aberration rate (7.0 % aberrant cells, exclusive gaps) was statistically signifìcant and biologically relevant increased after 4 hrs treatment with 262.5 µg/ml as compared to the corresponding solvent control (1.5% aberrant cells, exclusive gaps). ln addition, a dose related increase of the number of cells carrying structural chromosomal aberrations (1.0, 2.5, and 7.0 %) was observed at the concentration range evaluated (87.5 to 2625 µg/ml ). The observation of distinct increased numbers of micronucleated cells and of cells containing fragmented nuclei after 4hrs treatment with 262.5 µg/ml give additional evidence for a genotoxic potential of the test item. Therefore, these findings have to be regarded as being biologically relevant.

ln both experiments, EMS (100 and 200 µg/ml, respectively) and CPA (0.7 and 1.0 µg/ml, respectively) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Any other information on results incl. tables

Experiment	Fixation interval	Test item concentration in µg/ml	Polyploid cells in %	Cell numbers in % of control	Mitotic indices in % of control	Aberrant cells
						Inc. gaps*	Excl. gaps*	With exchanges
Exposure period 4 hrs without S9 mix
I	18h	negative control	2.2	n.t.	100	2.0	1.5	0.5
		solvent control^1	2.3	100	100	1.0	0.5	0.0
		positive control^3	2.3	n.t.	98	17.5	13.5 ^S	7.5
		175.0	2.6	76	100	1.5	1.5	0.0
		262.5	1.8	59	63	5.0	3.0 ^S	2.5
		350.0	2.0	47	81	2.0	0.5	0.0
Exposure period 18 hrs without S9 mix
I	18h	negative control	1.8	n.t.	100	2.0	0.5	0.0
		solvent control^1	2.2	100	100	1.0	0.0	0.0
		positive control^2	3.4	n.t.	101	11.0	9.0 ^S	4.0
		175.0	4.0	87	103	0.5	0.0	0.0
		262.5	2.0	83	106	1.5	0.5	0.5
		350.0	2.7	67	83	1.5	0.5	0.0
Exposure period 28 hrs without S9 mix
II	28h	negative control	1.7	n.t.	100	1.0	1.0	0.5
		solvent control^1	1.7	100	100	3.5	2.5	0.5
		positive control^2	1.4	n.t.	102	11.5	10.0 ^S	2.0
		100.0	2.1	77	71	3.0	2.0	0.0
Exposure period 4hrs with S9 mix
I	18h	negative control	2.1	n.t.	100	1.5	1.0	0.5
		solvent control^1	1.6	100	100	2.0	1.5	0.5
		positive control^2	1.5	n.t.	101	12.0	9.0 ^S	4.0
		43.8	2.9	112	75	1.0	1.0	0.5
		87.5	2.1	119	96	2.0	1.0	0.0
		175.0	1.7	82	58	2.0	1.5	0.0
II	28h	negative control	1.9	n.t.	100	1.5	1.5	0.0
		solvent control^1	2.2	100	100	1.5	1.5	0.0
		positive control^3	1.2	n.t.	98	11.5	9.5 ^S	4.5
		87.5	2.4	113	87	2.0	1.0	0.0
		175.0	2.6	64	86	4.5	2.5	0.5
		262.5	3.3	42	52	11.5	7.0 ^S	1.0

*inclusive cells carrying exchanges

n.t. not tested

^Saberration frequencystatistically significant higher than corresponding control values

¹deionised water 10% (v/v)

²EMS 100 µg/ml

³EMS 200 µg/ml

Applicant's summary and conclusion

Conclusions:

It can be stated that under the experimental conditions reported, the test item induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.
Therefore, RED Rwa 4565 is considered to be clastogenic in this chromosome aberration test in the presence of S9 mix at high toxicity level.

Executive summary:

The test item RED Rwa 4565, dissolved in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. The following study design was performed:

	Without S9 mix			With S9 mix
	Exp. I	Exp. II		Exp. I	Exp. II
Exposure period	4 hrs	18 hrs	28 hrs	4 hrs	4 hrs
Recovery	14 hrs	-	-	14 hrs	24 hrs
Preparation interval	18 hrs	18 hrs	28 hrs	18 hrs	28 hrs

ln each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations.

The highest applied concentration in the pre-test on toxicity (5310 pg/ml) was chosen respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiments was performed considering the toxicity data. The chosen treatment concentrations are described in Table 2, Page 17 of the study report. The evaluated experimental points and the results are summarised in Table 1, page 10 of the study report.

Toxic effects indicated by reduced cell numbers of below 50 % of control were observed in experiment I in the absence of S9 mix and in experiment ll in the presence of S9 mix. No toxic effects indicated by reduced mitotic indices were observed at the test item concentrations evaluated. However, concentrations showing clearly reduced mitotic indices were not evaluable for cytogenetic damage.

ln experiment ll, in the presence of S9 mix, at the 28 hrs preparation interval a statistically significant and biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed at the highest test item concentration evaluated.

No increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.