[x·sodium, y·lithium, x+y=3] 4-amino-3[[4-[[4-[(2-amino-4-hydroxyphenyl)diazenyl]phenyl]amino]-3-sulfonato phenyl]diazenyl]-5-hydroxy-6-(phenydiazenyl)naphthalene-2,7-disulfonat, mixture of isomers

Genetic toxicity: in vitro

Administrative data

Endpoint:

genetic toxicity in vitro, other

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

See the report for the analogue approach justification in section 13 "Other reports"

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From November 17 to December 23, 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted May 26th, 1983

Qualifier:

according to guideline

Guideline:

other: EU Method B.14

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA1538, TA 98 and TA 100

Details on mammalian cell type (if applicable):

- Periodically checked regular checking of the properties of the strains with regard to membrane permeability and ampicillin resistance as well as normal spontaneous mutation rates is performed in testing laboratory according to Ames et al.
- Storage: the strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.
- Precultures: from the thawed ampoules of the strains 0.5 ml bacterial suspension was transferred to 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. This nutrient medium contains per litre: 8 g Difco Nutrient Broth, 5 g NaCl.
- Incubation: the bacterial culture was incubated in a shaking water bath for 6 hours at 37° C.

Metabolic activation:

with and without

Metabolic activation system:

homogenate of rat liver (S9), induced by Arochlor 1254

Test concentrations with justification for top dose:

10.0, 100.0, 333.3, 1000.0 and 5000.0 μg/plate in both experiments

Vehicle / solvent:

- Solvents used: sterile, redistilled water (for the test substance and sodium azide reference substance solution) and DMSO (for 2-aminoanthracene and 4-nitro-o-phenylendiamine reference solutions).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylenediamine // 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation).
- Selective Agar: 2.0 % Vogel-Bonner-Glucose-Minimal-Agar was used as selective agar. Each petri dish was filled with 20 ml of this nutrient medium. Sterilizations were performed at 121° C in an autoclave.
- Overlay Agar: the overlay agar contains per litre 6.0 g Difco Bacto Agar, 6.0 g NaCl, 10.5 mg L-histidine x HCl x H2O, 12.2 mg biotin. Sterilizations were performed at 121° C in an autoclave.

NUMBER OF REPLICATIONS: for each strain and dose level, including the controls, a minimum of three plates were used.

SETTING UP THE PLATES: 0.1 ml of test solution at each dose level, solvent control, negative control, or reference mutagen solution (positive control) and 0.5 ml of S9 mix (for test with metabolic activation) or S9 mix substitution-buffer (for test without metabolic activation) and 0.1 ml of bacteria suspension and 2 ml of overlay agar were mixed in a test tube and poured onto the selective agar plates. After solidification the plates were incubated upside down for 72 hours at 37 °C in the dark.

PRE EXPERIMENT FOR TOXICITY AND DOSE SELECTION: prestudy was performed with strains TA 98 and TA 100. 8 concentrations were tested for toxicity and mutation induction with each 3 plates in the same conditions as for the main experiment. Toxicity of the test article may be evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures.

DATA RECORDING: the colonies were counted using the BIOTRAN III counter. If precipitation of the test article precluded automatic counting the revertant colonies were counted by hand.

PREPARATION OF MAMMALIAN MICROSOMAL FRACTION S9 MIX
- S9: the S9 liver microsomal fraction was obtained from the liver of 8-12 weeks old male Wistar rats, strain WU which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 in olive oil 5 days previously. After cervical dislocation the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate, diluted 1:3 in KCl was centrifuged cold at 9.000 g for 10 minutes. A stock of the supernatant containing the microsomes was frozen in ampoules of 2 or 5 ml and stored at - 70 °C. Small numbers of the ampoules were kept at - 20 °C for only several weeks before use. The protein concentration in the S9 preparation is usually between 20 and 45 mg/ml.
- S9 mix: before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution in a ratio 3:7. The composition of the cofactor solution was concentrated to yield the concentrations in the S9 mix: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 5 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. During the experiment the S9 mix was stored in an ice bath.

Evaluation criteria:

For the evaluation of the results the corresponding background growth on both negative vontrol and test plates and the normal range of spontaneous reversion rates are used.
A test article is considered as positive if either a significant dose-related increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A test article producing neither a significant dose-related increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
A significant response is described as follows: a test article is considered as mutagen if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, TA 1538, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Statistics:

No evaluated statistical procedure was used.

Species / strain:

S. typhimurium TA 1538

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

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA 1535, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in TA1535, at 100.0 μg/plate (exp 1) without metabolic activation and in TA98 at 1000 and 5000 μg/plate with metabolic activation (exp. I & exp.II)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Toxic effects evidenced by a reduction in the number of spontaneous revertants occurred in strain TA 1535 at 100.0 µg/plate (exp. I), and in strain TA 1537 at 1000.0 µg/plate (exp. Ill) both without S9 mix. Also in strain TA 98 at 5000.0 µg/plate with metabolic activation in both experiments.
The plates incubated with the test article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all strains used.
A slight increase in revertant colony numbers was observed in strain TA 100 (exp. I) at 333.3 µg/plate. This effect was not reproduced in the independent experiment and therefore it is considered not to be relevant.
In strain TA 1537 (with S9 mix) a dose-dependent increase in the number of revertants was observed in the concentration range of 333.3 up to 5000.0 µg/plate. This increase was less significant in exp. II. To prove the effects of exp. I and exp. II a third experiment was performed. In this additional experiment no relevant increase in the number of revertants was observed. Based on these results a mutagenic potential of the test article in strain TA 1537 cannot be excluded, however the effect was not reproduced in all three experiments.
In strain TA 1538 (with S9 mix) in both experiments the numbers of revertants were distinctly enhanced at concentrations higher than 10.0 µg/plate. In exp. I a dose-dependent increase was obtained up to 333.3 ug/plate and in exp. II up to 1000.0 µg/plate.
At higher concentrations a decrease in the number of revertants was obtained indicating beginning of toxicity.
Up to the highest investigated dose, neither a significant and reproducible increase of the number of revertants was found in the strains TA 1535, TA 98, and TA 100 as compared to the solvent control nor a concentration-dependent enhancement of the revertant number exists. The presence of liver microsomal activation did not influence these findings.
Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced revertant colonies.

The following table presents the revertants/plate mean for the three experiments for each strain.

	Revertants/plate mean from three plates, without S9 mix
Dose μg/plate I/II/III	TA1535			TA1537			TA1538		TA98		TA100
	I	II	III	I	II	III	I	II	I	II	I	II
Neg. Control	9	7	5	4	6	6	15	14	20	17	114	77
Solv. Control	9	6	8	4	4	6	15	13	18	16	110	71
10.0	7	6	9	5	5	5	14	18	18	20	112	60
100.0	4	8	9	4	5	4	14	12	21	22	113	61
333.3	7	6	7	6	5	4	12	18	25	18	100	69
1000.0	5	4	5	4	4	3	14	23	25	16	86	64
5000.0	6	4	5	7	4	6	17	18	21	16	98	57
Sodium azide (10 μg/plate)	697	905	579
4-Nitropophenylene- diamine (50 μg/plate)				171	155	149	1126	1148	1895	1230
	Revertants/plate mean from three plates, with S9 mix
Dose μg/plate I/II/III	TA1535			TA1537			TA1538		TA98		TA100
	I	II	III	I	II	III	I	II	I	II	I	II
Neg. Control	5	7	5	4	4	5	18	17	44	19	116	84
Solv. Control	5	8	7	4	5	6	14	13	44	14	100	75
10.0	7	10	5	5	5	4	23	16	43	17	105	65
100.0	7	11	8	4	7	10	39	37	48	20	123	92
333.3	8	8	10	8	8	7	45	35	47	19	164	98
1000.0	6	9	8	10	5	5	25	55	28	8	101	75
5000.0	7	12	7	12	10	9	22	26	22	7	121	66
2-aminoanthracene (10 μg/plate)	82	97	540	179	319	175	1867	1563	2455	2231	2285	1496

Conclusions:

Under the tested conditions, the test article induced point mutations by frameshifts in the genome of strain TA1538. There was also an indication of a possible mutagenic response in strain TA1537.

Executive summary:

The substance was assessed for its potential to induce gene mutations according to the plate incorporation test using Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100. The assay was performed in two independent experiments, using identical procedures, both with and without rat liver microsomal activation. According to the results of these experiments, a third experiment (III) was performed only with TA1535 and TA1537 with and without metabolic activation. Five concentrations (10.0, 100.0, 333.3, 1000.0 and 5000.0 μg/plate) including the control were inoculated with each of the strains for 72 hours at 37 °C. Each concentration was tested in triplicate.

Toxic effects occurred in strain TA1535 at 100.0 μg/plate (exp. I) and in strain TA1537 at 1000.0 μg/plate (exp. Ill) both without S9 mix. Also in strain TA 98 at 5000.0 μg/plate with metabolic activation in both experiments. The plates incubated with the test article showed normal background growth up to 5000.0 ug/plate with and without S9 mix in all strains used. In strain TA1537 (with S9 mix) a dose-dependent increase in the number of revertants was observed in the concentration range of 333.3 up to 5000.0 ug/plate. This increase was less significant in exp. II. In the experiment III no relevant increase in the number of revertants was observed. Based on these results a mutagenic potential of the test article in strain TA1537 cannot be excluded, but this the effect was not reproduced in all three experiments. In strain TA1538 (with S9 mix) a dose-dependent increase was obtained up to 333.3 μg/plate (exp. I) and up to 1000.0 μg/plate (exp. ΙI). No indication of mutagenic response occured in strains TA1535, TA 98, and TA 100 as compared to the solvent control nor a concentration-dependent enhancement of the revertant number exists, with and without metabolic activation. The substance induced point mutations by frameshifts in the genome of the strain TA1538 and there is an indication of a possible mutagenic response in strain TA1537.

Conclusion

Under the tested conditions, the test article induced point mutations by frameshifts in the genome of strain TA1538. There was also an indication of a possible mutagenic response in strain TA1537.

Cross-reference 2

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From October 31, 1990 to April 12, 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted May 26th, 1983

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

other: in vitro chromosome aberration in mammalian cells

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: LMP, D-6100 Darmstadt.
- Doubling time: 12-16 hrs in stock culture.
- Plating efficiency: more than 50 %.
- Methods for maintenance in cell culture: stored in liquid nitrogen.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: seeding was done with about 5 x 10^5 cells per flask in 15 ml of MEM (minimal essential medium) supplemented with 10 % fetal calf serum. The cells were subcultured twice weekly. The cell cultures were incubated at 37 °C and 4.5 % CO2 atmosphere.
- Properly maintained: yes. Stored in liquid nitrogen.
- Periodically checked for mycoplasma contamination: yes. Before freezing.
- Periodically checked for karyotype stability: yes. Before freezing.

Metabolic activation:

with and without

Metabolic activation system:

homogenate of rat liver (S9), induced by Arochlor 1254.

Test concentrations with justification for top dose:

Without S9 mix
- 7 h: 0.010; 0.030; 0.100; 0.300 mg/ml.
- 18 h: 0.001; 0.003; 0.010; 0.030; 0.100; 0.300 mg/ml.
- 28 h: 0.010; 0.030; 0.100; 0.300 mg/ml.
With S9 mix
- 7 h: 0.030; 0.100; 0.300; 1.000 mg/ml.
- 18 h: 0.003; 0.010; 0.030; 0.100; 0.300; 1.000 mg/ml.
- 28 h: 0.030; 0.100; 0.300; 1.000 mg/ml.

Vehicle / solvent:

- Solvent used: saline G was used to dissolve the test article. One litre of the solvent was composed of 8000 mg NaCl, 400 mg KCl, 1100 mg Glucose, 290 mg Na2HP04.7H20, 150 mg KH2P04 and the pH was adjusted to 7.2
- Justification for choice of solvent: it was chosen according to its solubility properties and its non-toxicity for the cells.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

PRE EXPERIMENT FOR TOXICITY AND DOSE SELECTION
In order to establish a concentration dependent plating efficiency relationship a pre-experiment was performed in the same conditions as for the main experiment. 8 concentrations were tested (0.001; 0.010; 0.030; 0.100; 0.300; 1.000; 2.000; 4000 mg/ml). Per concentration duplicate cultures were used. The mitotic index was determined in 1000 cells from each of the two slides per test group. Toxicity of the test article was evidenced by a reduction in plating efficiency. The highest dose level used was 10 mM unless limited by the solubility of the test article or that producing some indication of cytotoxicity (reduced plating efficiency and/or partial inhibition of mitosis). If toxic effects were produced the highest dose level should reduce the plating efficiency to approximately 20 - 50 %. In addition, this concentration should suppress if possible mitotic activity (% cells in mitosis) by approximately 50 %, but not so great a reduction that insufficient scorable mitotic cells can be found. According to the results of the pre-experiment six concentrations (18 h interval) were selected for the chromosomal aberration assay.

SEEDING OF THE CULTURES
Two days old logarithmically growing stock cultures more than 50 % confluent were trypsinised 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. One litre of Ca-Mg-free salt solution was composed as follows of 8000 mg NaCl, 400 mg KCl, 1100 mg glucose, 350 mg NaHC03. Prior to trypsin treatment the cells were rinsed with the Ca-Mg-free salt solution containing 200 mg/ml EDTA (Ethylene diamine tetraacetic acid). Then the cells were seeded into Quadriperm dishes which contained microscopic slides (2 chambers per dish and test group). In each chamber 5 x 10 ^4 - 1 x 10^5 cells were seeded with regard to preparation time. The medium was MEM + 10 % FCS (complete medium).

TREATMENT OF THE CULTURESA
fter 48 h (7 h, 28 h preparation interval) and 55 h (18 h preparation interval) the medium was replaced with serum-free medium containing the test article, either without S9 mix or with 20 μl/ml S9 mix. Concurrent negative and positive controls (with and without S9 mix) were performed in parallel. After 4 h this medium was replaced with complete medium after rinsing twice with saline G. All incubations were done at 37° C in a humidified atmosphere with 4.5 % CO2.

CHROMOSOME PREPARATION
5, 15.5 and 25.5 h after the start of the treatment colcemid was added (0.2 μg/ml culture medium) to the cultures. 2.0 h (7 h interval) or 2.5 h later, (18 h and 28 h interval) the cells were treated on the slides 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 3:1 absolute methanol:glacial acetic acid. Both slides per group were prepared. After fixation the cells were stained with giemsa.

ANALYSIS OF METAPHASE CELLS
The evaluation of the slides was performed using NIKON microscopes with 100 x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well spread metaphases per slide were scored for cytogenetic damage on coded slides except in the positive control samples where 25 metaphases were scored. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To determine a cytotoxic effect the mitotic index was determined.

OTHER EXAMINATIONS:
- Determination of polyploidy: the number of polyploid cells (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype) was scored.

PREPARATION OF MAMMALIAN MICROSOMAL FRACTION S9 MIX
- S9: the S9 liver microsomal fraction was obtained from the liver of 8-12 weeks old male Wistar rats, strain WU which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 in olive oil 5 days previously. After cervical dislocation the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate, diluted 1:3 in KCl was centrifuged cold at 9.000 g for 10 minutes. A stock of the supernatant containing the microsomes was frozen in ampoules of 2 or 3 ml and stored at - 70 °C. Small numbers of the ampoules were kept at - 20 °C for only several weeks before use. The protein concentration in the S9 preparation is usually between 20 and 45 mg/ml, the S9 used in this study had 30.3 mg/ml protein concentration.
- S9 mix: on the day of the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.3 mg/ml in the cultures. The composition of the cofactor solution was concentrated to yield the concentrations in the S9 mix: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 5 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. During the experiment the S9 mix was stored in an ice bath.

ACCEPTABILITY OF THE ASSAY
1. The number of aberrations found in the negative and/or solvent controls fall within the laboratory historical control data range: 0.00 % - 4.00 %.
2. The positive control substances should produce significant increases in the number of cells with structural chromosome aberrations.

Rationale for test conditions:

V79 cell line was chosen as it is used for many years in in-vitro experiments with success. The high proliferation rate (doubling timne 12-16 hrs in stock cultures) and the high plating efficiency of untreated cells (more than 50 %), both necessary for the appropriate performance of the study reccomend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22.

Evaluation criteria:

A test article is classified as mutagenic if it induces either a significant dose-related increase in the number of structural chromosomal aberrations or a significant positive response for at least one of the test points. A test article producing neither a significant dose-related increase in the number of structural chromosomal aberrations nor a significant positive response at any one of the test points is considered non-mutagenic in this system.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the chi-square test. Evaluation was performed only for cells carrying aberrations exclusive gaps.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in conc. higher than 1 µg/ml (without S9 mix) and 30 µg/ml (with S9 mix)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES
In the pre-experiment for toxicity the colony forming ability of the V79 cells was distinctly reduced with concentrations higher than 0.001 (without S9 mix) and 0.030 mg/ml (with S9 mix). Based on the results of the pre-experiment and the cytogenic experiment, in the main experiment, in the absence of S9 mix cultures treated with 0.1 mg/ml (7 h interval) and 0.3 mg/ml (18 h and 28 h interval could be evaluated as top dose level. In the presence of S9 mix samples treated with 0.03 (interval 7 h) and 0.3 mg/ml (intervals 18 and 28 h) could be evaluated as top dose level. With higher concentrations not enough scorable cells could be found in the respective intervals. Treatment with concentrations higher than 0.001 mg/ml (without S9 mix) and 0.03 mg/ml (with S9 mix) reduced distinctly the plating efficiency of the V79 cells. Also, in the cytogenetic experiment the mitotic index was distinctly reduced in the samples treated with the highest scorable dose level at each fixation interval except at interval 7 (with S9 mix). However, at this test group at least a slight reduction was observed. One (7 h and 28 h) and three concentrations (18 h) were selected to evaluate metaphases for cytogenetic damage.

POLYPLOID METAPHASES
No relevant deviation from the control data was found after treatment with the test article. At fixation interval 18 h (with S9 mix), in the samples treated with 0.10 mg/ml the number of polyploid cells (8.0 %) is increased as compared to the corresponding solvent control (1.5 %). However, taking into account the polyploidy range of all the control groups in this study: 1.5 % - 12.0 % this value falls within that range and also in the range of the laboratory historical controls: 0 - 11 %.

POSITIVE CONTROL
EMS (0.72 mg/ml) and CPA (1.40 µg/ml) used as positive controls, showed distinct increases in cells with structural chromosome aberrations.

Both, in the absence and presence of S9 mix the test article did not increase the frequency of cells with aberrations at any fixation interval. The aberration rates of the cells after treatment with the test article (1.00 % - 5.00 %) were in or near to the range of the control values: 0.00 % - 4.50 %.

The mutagenicity data for each fixation interval are presented in the tables below.

Mutagenicity data at 7 h fixation interval.

Fixation interval 7 h
Article	number of cells analysed	conc. (mg/ ml)	S9 mix	per cent aberrant cells
				incl. Gaps	excl. Gaps	exchanges
Solvent-control	200	0.0	-	3.00	1.00	0.50
Test article	200	0.10	-	4.00	1.50	0.00
Solvent-control	200	0.0	+	2.50	1.00	0.00
Test article	200	0.03	+	6.50	3.00	0.00

Mutagenicity data at 18 h fixation interval.

Fixation interval 18 h after start of experiment
Article	number of cells analysed	conc. (mg/ ml)	S9 mix	per cent aberrant cells
				incl. Gaps	excl. Gaps	exchanges
Negative control	200	0.0	-	10.00	4.00	0.00
Solvent-control	200	0.0	-	6.50	3.50	1.50
Positive control EMS	50	0.72	-	22.00	16.00	12.00
Test article	200	0.01	-	1.50	1.00	0.00
Test article	200	0.03	-	4.00	2.00	0.50
Test article	200	0.10	-	2.50	1.00	0.00
Negative control	200	0.0	+	1.50	1.00	0.00
Solvent-control	200	0.0	+	6.50	4.50	0.50
Positive control EMS	25*	1.4	+	12.00	12.00	8.00
Test article	200	0.01	+	2.50	1.50	0.50
Test article	200	0.03	+	9.00	5.00	1.00
Test article	200	0.10	+	4.00	2.50	1.50

Mutagenicity data at 28 h fixation interval.

Fixation interval 28 h after start of the treatment
Article	number of cells analysed	conc. (mg/ ml)	S9 mix	per cent aberrant cells
				incl. Gaps	excl. Gaps	exchanges
Solvent-control	200	0.0	-	1.00	0.50	0.00
Test article	200	0.30	-	6.50	2.00	0.00
Solvent-control	100*	0.0	+	1.00	0.00	0.00
Test article	200	0.30	+	4.00	2.00	1.00

* one slide was not scorable; technical reasons

Conclusions:

The substance did not induce structural chromosome aberrations in the V79 Chinese hamster cell line.

Executive summary:

The substance was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in the absence and presence of metabolic activation, according to the OECD Guideline 473 and EU Method B10 . Prior to the main test the plating efficiency of the cells and the mitotic index were evaluated and the tested concentrations were chosen. The chromosomes were prepared at 7 h , 18 h and 28 h after the start of the treatment with the test article. The treatment interval was 4 h. In each experimental group two parallel cultures were used. 100 metaphases were scored for structural chromosome aberrations per culture; one (7 h and 28 h) and three concentrations (18 h) were selected to evaluate metaphases for cytogenetic damage.

Treatment with concentrations higher than 0.001 mg/ml (without S9 mix) and 0.03 mg/ml (with S9 mix) reduced distinctly the plating efficiency of the V79 cells. In the cytogenetic experiment the mitotic index was reduced in the samples treated with the highest scorable dose level at each fixation interval except at interval 7 (with S9 mix). With higher dose levels not enough scorable cells could be found. Both, in the absence and presence of S9 mix the test article did not increase the frequency of cells with aberrations at any fixation interval. The aberration rates of the cells after treatment with the test article (1.00 % - 5.00 %) were in or near the range of the control values: 0.00 % - 4.50 %.

Conclusion

The substance did not induce structural chromosome aberrations in the V79 Chinese hamster cell line, under the test conditions.

Cross-reference 3

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

November from 16 to 29, 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

other: EU Method B.14

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Bacteria growth: several days before the beginning of the study, 4 tials of the bacterial strains were defrosted and grown on Petri dishes with nutrient agar in order to obtain pure cultures.
- Storage: the plates were stored at 4 °C until required.
- Preparation: 16 hours before the study began, an inoculum of each of the four bacterial strains was prepared in 20 ml of nutrient broth and incubated at 37 °C. - Periodically checked: the histidine requirements, the presence or absence of plasmide pKM 101, the rfa factor (sensitivity to crystal violet) and the uvrβ factor (sensitivity to ultraviolet rays) were checked in the strains used to prepare the inocula.

Metabolic activation:

with and without

Metabolic activation system:

homogenate of rat liver (S9), induced by Arochlor 1254, at 10 % in standard cofactors

Test concentrations with justification for top dose:

31.25, 625, 1250, 2500, 5000 and 10000 μg/plate, for both experiments.

Vehicle / solvent:

- Solvents used: sterile, redistilled water (for the test substance and sodium azide reference substance solution) and DMSO (for 9 aminoacridine, 4-nitro-o-phenylendiamin and 2-aminoanthracen reference solutions).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2-aminoanthracene // 4-nitro-o-phenylendiamin

Remarks:

with metabolic activation

Details on test system and experimental conditions:

The tHo experiments Here carried out on di f ferent days.

METHOD OF APPLICATION: preincubation and plate incorporation.

NUMBER OF REPLICATIONS: three replicates per concentration.

SETTING UP OF THE TEST PLATES
- Plate preparation: 0.1 ml of bacterial culture and 0.1 ml of the diluted test substance were added to 0.5 ml of phosphate buffer (without metabolic activation) or 0.5 ml of S-9 (with metabolic activation). For the controls, 0.1 ml of the corresponding solvents were added to each product.
- First incubation: plates were incubated for 20 minutes at 37 °C and added to 2 ml of top agar melted at 45 °C containing biotin and histidine.
- Incubation: the test tubes were rapidly mixed in a rotamixer, and poured on to plates containing a base layer of minimal agar. The agar added was left to solidify and the plates were incubated at 30 - 35 °C for 48 hours.

Statistics:

The results from the standard products and the control group were compared using the Student's t test. In each group, the statistical comparison between the different concentrations of the test substance was carried out using one-way analysis of the variance.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Additional information on results:

The substance showed no toxicity for any of the strains at any of the concentrations tested, in both experiments I and II.
In experiment I, no mutagenic response was observed for any of the strains tested, with or without the addition of S9; strain TA98 showed a statistically significant response but it can not be considered to be mutagenic as the highest revertants count never doubled that of the control.
During the experiment II, no mutagenic response was observed for any of the strains tested, with or without the addition of S9.

POSITIVE CONTROLS
In both experiments I and II, all the bacterial strains responded positively and the metabolic activation system responded as expected.
Aminoanthracen, at a concentration of 10 µg/ plate, showed toxicity in strain TA1537 with the addition of S9, in the experiment I. In the experiment II, aminoanthracen, at a concentration of 10 µg/ plate, showed toxicity in strains TA1535 and TA1537 with the addition of S9.

Summary of results obtained in experiment 1

Bacterial  strain	% S-9	Mean no. of revertants per plate (μg/plate)
		0	31.25	62.5	125	250	500	1000
TA-1535	0	6.3	6.0	5.7	5.3	7.7	5.3	6.0
TA-1537	0	10.7	9.7	9.0	10.0	9.7	9.3	10.0
TA-98	0	14.0	12.0	14.3	12.3	14.0	14.3	14.0
TA-100	0	121.0	119.0	119.7	121.7	124.0	122.0	119.0
TA-1535	10	7.7	8.0	8.0	8.3	6.7	8.0	6.7
TA-1537	10	10.7	10.3	10.7	12.0	10.7	9.0	8.3
TA-98	10	21.7	22.7	22.3	25.7	28.0	29.0	20.3
TA-100	10	131.7	132.0	130.7	131.7	132.0	131.3	131.7

Summary of results obtained in experiment 2

Bacterial  strain	% S-9	Mean no. of revertants per plate (μg/plate)
		0	31.25	62.5	125	250	500	1000
TA-1535	0	13.7	12.0	14.0	13.7	13.3	12.7	13.3
TA-1537	0	12.0	13.3	11.3	12.0	13.0	10.7	12.3
TA-98	0	28.0	27.3	27.3	27.0	26.3	26.7	26.3
TA-100	0	150.0	151.0	147.3	148.0	146.0	149.0	148.0
TA-1535	10	13.7	13.0	13.7	13.3	14.0	14.3	14.3
TA-1537	10	18.0	17.0	16.7	18.3	17.7	19.7	16.7
TA-98	10	33.3	30.7	33.3	31.3	31.0	32.7	28.7
TA-100	10	150.7	148.3	151.0	150.0	150.0	147.3	150.3

Summary of statistical analysis for each strain

Experiment 1
Bacterial  strain	% S-9	Statistic F	Degrees of freedom		Significance of F (p)
			N	D
TA-1535	0	0.99	6	14	N.S.
TA-1537	0	0.58	6	14	N.S.
TA-98	0	1.67	6	14	N.S.
TA-100	0	2.23	6	14	N.S.
TA-1535	10	0.37	6	14	N.S.
TA-1537	10	2.44	6	14	N.S.
TA-98	10	4.53	6	14	V.S.
TA-100	10	0.10	6	14	N.S.
Experiment 2
Bacterial  strain	% S-9	Statistic F	Degrees of freedom		Significance of F (p)
			N	D
TA-1535	0	0.72	6	14	N.S.
TA-1537	0	0.41	6	14	N.S.
TA-98	0	0.23	6	14	N.S.
TA-100	0	0.90	6	14	N.S.
TA-1535	10	0.10	6	14	N.S.
TA-1537	10	1.18	6	14	N.S.
TA-98	10	0.92	6	14	N.S.
TA-100	10	0.36	6	14	N.S.

N.S. = Non-significant.

V.S = Significant for p < 0.01.

Conclusions:

The substance is non mutagenic to Salmonella strains, with and without metabolic activation.

Executive summary:

The mutagenic potential of the substance, using four strains of Salmonella typhimurium (TA 98, TA100, TA1535, TA1537), with and without the addition of a metabolic activation system, was assessed according to OECD Guideline 471 and EU Method B.14. Six concentrations of the substance (1000 - 500 - 250 - 125 - 62.5 and 31.25 μg/plate) were inoculated with each of the strains for 48 hours at 30 -35 °C in two experiments. Concurrent positive controls were used in order to assess the metabolic activation system, while solvent controls also run in parallel.

The substance showed no toxicity for any of the strains at any of the concentrations tested, in both experiments I and II.

In experiment I, no mutagenic response was observed for any of the strains tested, with or without the addition of S9; strain TA98 showed a statistically significant response but it can not be considered to be mutagenic as the highest revertants count never doubled that of the control.

During the experiment II, no mutagenic response was observed for any of the strains tested, with or without the addition of S9.

Conclusion

The substance is non mutagenic to Salmonella strains, with and without metabolic activation.

Materials and methods

Test material

Test material information

1

Reference substance name:

[x·sodium, y·lithium, x+y=3] 4-amino-3[[4-[[4-[(2-amino-4-hydroxyphenyl)diazenyl]phenyl]amino]-3-sulfonato phenyl]diazenyl]-5-hydroxy-6-(phenydiazenyl)naphthalene-2,7-disulfonat, mixture of isomers

EC Number:

827-162-2

Cas Number:

2218502-12-4

Molecular formula:

C34H24N9O11S3.NaxLiy

IUPAC Name:

[x·sodium, y·lithium, x+y=3] 4-amino-3[[4-[[4-[(2-amino-4-hydroxyphenyl)diazenyl]phenyl]amino]-3-sulfonato phenyl]diazenyl]-5-hydroxy-6-(phenydiazenyl)naphthalene-2,7-disulfonat, mixture of isomers

Test material form:

solid

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In conc. higher tan 1 microg/ml (without S9 mix) and 30 microg/ml (with S9 mix)

Test results 2

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 3

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 5

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 6

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity