disodium 7-(4,6-difluoropyrimidin-2-ylamino)-4-hydroxy-3-(4-methoxy-2-sulfonatophenylazo)naphthalene-2-sulfonate; reaction mass of: disodium 7-(2,4-difluoropyrimidin-6-ylamino)-4-hydroxy-3-(4-methoxy-2-sulfonatophenylazo)naphthalene-2-sulfonate

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 Protocol : October 17, 1996 - Date of Final Report : March 12, 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Title:

Unnamed

Year:

1996

Report date:

1997

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 May 26, 1983

Test guideline 2

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

enacted July 13, 1974, amended December 05, 1986

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EEC Directive 92/69, L 383 A, Annexe V, B 10

Version / remarks:

dated December 29, 1992.

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:

426-840-1

EC Name:

-

IUPAC Name:

tetrasodium 7-[(2,6-difluoropyrimidin-4-yl)amino]-4-hydroxy-3-[2-(4-methoxy-2-sulfophenyl)diazen-1-yl]naphthalene-2-sulfonate 7-[(4,6-difluoropyrimidin-2-yl)amino]-4-hydroxy-3-[2-(4-methoxy-2-sulfophenyl)diazen-1-yl]naphthalene-2-sulfonate

Test material form:

solid

Details on test material:

Identity: Scarlet RN 1165
Appearance : Solid powder, dark-red

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, D-64287 Darmstadt) were stored in liquid nitrogen in the cell bank of C C R allowing the repeated use of the same cell culture batch in experiments

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 applicable concentration range of the test article for the cytogenetic experiments was determined in a pre-test using the determination of cell numbers 24 h after start of treatment as indicator for toxicity response. The highest concentration used in the pre-test on toxicity (5000 μg/ml) was chosen according to the current OECD Guideline for in vitro mammalian cytogenetic tests. Test article concentrations between 3 and 5000 μg/ml (with and without S9 mix) were chosen for the examination of the cytotoxic potential. In the absence of S9 mix toxic effects indicated by a strong reduction of the cell number (< 30 % of control) were observed after treatment with 1000 μg/ml and above, whereas in the presence of S9 mix no toxic effects could be observed.
In experiment I, test article concentrations within a range from 10 - 500 μg/ml (without S9 mix) or 30 - 5000 μg/ml (with S9 mix) were applied for investigation of the potential to induce cytogenetic damage.
In experiment II the investigated concentration range was 30 - 400 μg/ml (without S9 mix) or 30 - 1000 μg/ml (with S9 mix).
In the pre-test and both main experiments, without and with S9 mix, precipitation of the test article in culture medium occurred at concentrations from 300 μg/ml and above.

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

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent solvent controls (MEM)

True negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

EXPERIMENTAL PERFORMANCE

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 enzymatic 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: Difeo Laboratories, Detroit, USA).The Ca-Mg-free salt solution was composed as follows (per litre): NaCl 8000 mg, KCl 400 mg, Glucose 1000 mg, NaHCO3 350 mg.
Prior to the trypsin treatment the cells were rinsed with Ca-Mg-free salt solution containing 200 mg/I 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). In each chamber 1 x 10EXP4
6 x 10EXP4 cells were seeded with regard to preparation time. The medium was MEM +10 % FCS ( complete medium).

Treatment:
Exposure time 4 hours (with 89 mix):
After approximately 71.5 h (exp. I) or 47.5 h (exp. II) at the 28 h preparation interval and approximately 75 .5 (exp. I) or 54.5 (exp. II) at the 18 h preparation interval the culture medium was replaced with serum-free medium containing different concentrations of the test article and 50 μI/ml S9 mix.
After 4 h 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 and KH2PO4 150 mg. pH was adjusted to 7.2.

Exposure time 18 and 28 hours (without S9 mix):
After approximately 71.5 h (exp. I) or 47.5 h (exp. 11) at the 28 h preparation interval and approximately 75.5 (exp. I) or 54.5 (exp. II) at the 18 h preparation interval the culture medium was replaced with complete medium (10 % FCS) containing different
concentrations of the test article without 89 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 % C02 (95 .5 % air).

Preparation of the Cultures:
15.5 and 25.5 h after the start of the treatment colcemid was added (0.2 μg/ml culture medium) to the cultures. 2.5 h later, the cells on the slides were treated in the chambers with hypotonic solution (0.4 % KC]) for 20 min at 37° C. After incubation in the hypotonic solution the cells were fixed with 3 + 1 methanol + glacial acetic acid. Per experiment both slides per group were prepared. After preparation the ceJls were stained with Giemsa (E. Merck, D-64293 Darmstadt).
AdditionaJly, two cultures per treatment group, not treated with Colcemid, were set up in parallel. These cultures were stained in order to determine microscopicaJly the cell number within 10 defined fields per slide. The toxicity of the substance is given as reduction of % cells as compared to the negative control.

Analysis of Metaphase Cells:
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, 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. I 00 well spread metaphases 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 was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).

Acceptability of the assay:
The chromosome aberration assay 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. 00 % - 4. 00 %.
b) The positive control substances should produce significant increases of the number of cells with structural chromosome aberrations.

Evaluation criteria:

A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups are 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 are 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.

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. If 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.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criteria is valid:

A test item can be classified as aneugenic if:
- the number of induced numerical aberrations are not in the range of our historical control data (0.0 % - 8.5 % polyploid cells).

Statistics:

Statistical significance was confirmed by means of the Fischer's exact test (10). However, both biological and statistical significance should be considered together.
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

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

In the pre-test on toxicity in the presence of S9 mix the test article did not induce cytotoxicity. Without S9 mix a strong reduction of the cell numbers (< 30 % of control) after treatment with 1000 μg/ml and higher concentrations was observed.
In the pre-test and both main experiments, without and with S9 mix, precipitation of the test article in culture medium occurred at concentrations from 300 μg/ml and above.

In both cytogenetic experiments, in the presence of S9 mix cultures after treatment with 1000 μg/ml (18 h and 28 h) were evaluated for cytogenetic damage. In the absence of S9 mix cultures after treatment with 300 μg/ml as maximum concentration could be evaluated in all experiments. At higher concentrations, in the absence and the presence of S9 mix, heavy precipitation and strong adhesion of the test article to the slides and the surface of the cells occurred, interfering with the preparation and the evaluation of the chromosomes. Therefore evaluation of cytogenetic damage was not feasible at higher concentrations. In addition, in the absence of S9 mix, strong toxicity hindered the evalation of cultures after treatment with the concentrations > 300 μg/ml.

In the absence as well as in the presence of S9 mix, in both experiments the mitotic indices were not reduced after treatment with the highest evaluated concentrations. Determination of cell numbers revealed a strong reduction in the absence of 89 mix after treatment with 300 μg/ml in experiment I only (46.4 %).

In both experiments, in the absence and presence of S9 mix the test article did not increase the frequency of cells with aberrations. The aberration rates of the cells after treatment with the test article (exp.I and II: 0.0 % - 3.0; exclusive gaps) were in or near to the range of the solvent control values (exp. I: 1.0 % - 2.0 %; exp. II: 1.0 % - 4.0 %) and in the range of our historical control data: 0.0 % - 4.0 %.

In both experiments, EMS (600 μg/ml) and CPA (0.71 μg/ml) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Any other information on results incl. tables

Summary of results of the chromosome aberration study with SCARLET RN 1165:

Experiment	S9 mix	Fixation interval	Test item concentration in µg/ml	Polyploid cells in %	Mitotic index in % of control	Aberrant cells
						Inc. gaps	Excl. gaps	Exchanges
I	-	18h	Solvent control	3.5	100.0	2.5	2.0	0.0
	-		30.0	5.0	11.5	3.0	2.5	0.5
	-		100.0	2.5	109.0	1.5	1.0	0.5
	-		300.0^P	1.0	102.8	1.0	0.0	0.0
II	-	18h	Solvent control	5.0	100.0	1.5	1.0	0.0
	-		30.0	2.0	78.6	1.0	0.5	0.0
	-		50.0	1.5	99.3	2.0	1.0	0.0
	-		300.0^P	3.0	89.4	0.5	0.5	0.5
I	-	28h	Solvent control	2.5	100.0	2.0	2.0	0.5
	-		100.0	2.5	107.0	0.0	0.0	0.0
	-		300.0^P	2.0	99.2	1.0	0.5	0.0
II	-	28h	Solvent control	4.5	100.0	5.0	4.0	0.0
	-		200.0	3.5	93.3	3.5	3.0	0.0
	-		300.0^P	1.0	108.7	1.0	0.0	0.0
I	+	18h	Solvent control	3.5	100.0	2.0	1.0	0.0
	+		30.0	1.5	97.3	1.0	0.5	0.0
	+		100.0	3.0	95.6	3.5	3.0	1.0
	+		300.0^P	3.5	100.3	3.5	2.0	0.0
	+		1000.0^P	3.0	110.3	2.0	1.5	1.0
II	+	18h	Solvent control	2.0	100.0	4.0	3.5	2.5
	+		30.0	3.5	109.5	4.0	3.0	1.0
	+		100.0	2.0	80.7	2.5	2.0	0.0
	+		300.0^P	4.5	92.6	1.5	0.5	0.0
	+		1000.0^P	4.0	87.8	4.5	2.5	0.5
I	+	28h	Solvent control	3.0	100.0	3.5	2.0	0.0
	+		100.0	1.5	104.7	0.0	0.0	0.0
	+		300.0^P	2.5	105.7	1.0	1.0	0.0
	+		1000.0^P	1.0	110.4	1.5	1.5	0.5
II	+	28h	Solvent control	1.5	100.0	3.0	1.0	0.0
	+		200.0	3.0	80.7	3.0	2.0	0.0
	+		300.0^P	4.5	83.8	2.5	2.5	0.0
	+		1000.0^P	3.0	77.2	3.5	1.5	0.0

*inclusive cells carrying exchanges

Aberrant cells in the positive control groups: 27.0%-31.5%

^PPrecipitation was observed

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, SCARLET RN 1165 is considered to be non-mutagenic in this chromosome aberration test.

Executive summary:

The test article SCARLET RN 1165, dissolved in culture medium (MEM), was assessed for its potential to induce structural chromosome aberrations in vitro in V79 cells of the Chinese hamster in two independent experiments. The chromosomes were prepared 18 h and 28 h after start of treatment with the test article. The treatment interval was 4 h with metabolic activation, 18 h and 28 h without metabolic activation. In each experimental group two parallel cultures were set up. Per culture 100 metaphases were scored for structural chromosome aberrations.

The applicable concentration range of the test article for the cytogenetic experiments was determined in a pre-test using the determination of cell numbers 24 h after start of treatment as indicator for toxicity response. The highest concentration used in the pre-test on toxicity (5000 μg/ml) was chosen according to the current OECD Guideline for in vitro mammalian cytogenetic tests. Test article concentrations between 3 and 5000 μg/ml (with and without S9 mix) were chosen for the examination of the cytotoxic potential. In the absence of S9 mix toxic effects indicated by a strong reduction of the cell number (< 30 % of control) were observed after treatment with 1000 μg/ml and above, whereas in the presence of S9 mix no toxic effects could be observed.

In experiment I, test article concentrations within a range from 10 - 500 μg/ml (without S9 mix) or 30 - 5000 μg/ml (with S9 mix) were applied for investigation of the potential to induce cytogenetic damage. In experiment II the investigated concentration range was 30 - 400 μg/ml (without S9 mix) or 30 - 1000 μg/ml (with 89 mix). The evaluated experimental points and the results are presented in table 1 (page 10).

In the pre-test and both main experiments, without and with S9 mix, precipitation of the test article in culture medium occurred at concentrations from 300 μg/ml and above.

In this study, at both preparation intervals, in the absence and the presence of S9 mix, no relevant reduction of the mitotic indices could be observed. Determination of cell numbers revealed a strong reduction in the absence of S9 mix after treatment with 300 μg/ml in experiment I only. However, evaluation of cultures after treatment with higher concentrations was not feasible, since either the test article covered the cells and the chromosomes or strong toxicity was observed.

In both independent experiments, there were no biologically relevant increases in cells carrying structural chromosome aberrations after treatment with the test article at preparation intervals 18 hand 28 h (with and without 89 mix).

In both experiments, no biologically relevant increase in the frequencies of polyploid metaphases was found after treatment with the test article 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.