Tetrasodium 3-[[4-[[4,6-bis-[(3-sulfonatoprop-1-yl)thio]-1,3,5-triazin-2-yl]amino]-2-methyl-5-methoxy-phenyl]azonaphthalene-1,5-disulfonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 August 1995 to 17 October 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to valid guidelines and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tetrasodium 3-[[4-[[4,6-bis-[(3-sulfonatoprop-1-yl)thio]-1,3,5-triazin-2-yl]amino]-2-methyl-5-methoxy-phenyl]azonaphthalene-1,5-disulfonate

EC Number:

421-950-6

EC Name:

Tetrasodium 3-[[4-[[4,6-bis-[(3-sulfonatoprop-1-yl)thio]-1,3,5-triazin-2-yl]amino]-2-methyl-5-methoxy-phenyl]azonaphthalene-1,5-disulfonate

Cas Number:

187674-70-0

Molecular formula:

C27H26N6Na4O13S6

IUPAC Name:

tetrasodium 3-{2-[4-({4,6-bis[(3-sulfonatopropyl)sulfanyl]-1,3,5-triazin-2-yl}amino)-5-methoxy-2-methylphenyl]diazen-1-yl}naphthalene-1,5-disulfonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Appearance: yellow-brown to orange powder
- Storage conditions of test material: at room temperature

Method

Target gene:

Histidine operon

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

- Type and identity of media: Cultures were stored in cryoprotective medium containing 1 mL nutrient broth and 0.09 mL dimethyl sulfoxide (DMSO) in a liquid nitrogen container.
- Culture preparation prior to study initiation: The day before treatment, cultures were inoculated from frozen permanents: a crystal was sampled under sterile conditions and put into approximately 6 mL of nutrient broth. The nutrient broth was then placed under agitation in an incubator at 37 °C for about 14 hours.

Additional strain / cell type characteristics:

other: rfa in all strains; uvr B in TA 1535, TA 100, TA 1537 and TA 98; R-factor pKM 101 in TA 98, TA 100 and TA 102; pAQ1 in TA 102.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Concentrations: 30, 100, 300, 1000 and 3000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

congo red

mitomycin C

other: 2-anthramine

Remarks:

The vehicle for the positive controls was DMSO for all substances except Mitomycin C which was dissolved in distilled water.

Details on test system and experimental conditions:

MAIN TEST: Two independent tests were run both with and without metabolic activation, following the methodology detailed below.

METHOD OF APPLICATION: Preincubation method; 0.05 to 0.1 mL of the test solution, 0.5 mL of phosphate buffer pH 7.4 (without S9 mix) or of S9 mix (with S9 mix) and 0.1 mL of the strain were incubated for 30 minutes at 30 °C before adding the overlay agar and pouring onto the surface of a minimum agar plate (the agar contained 0.5 % glucose).

DURATION
- Preincubation period: 30 minutes at 30 °C.
- Exposure duration: After preincubation cultures were incubated for a further 48 to 72 hours at 37 °C on the agar plates.

NUMBER OF REPLICATIONS: Three plates per concentration and for each of the controls.

SCORING: Revertants colonies were scored with an automatic counter (Artek counter, model 880, O.S.I., 75015 Paris, France). During each test, for each strain and for each experimental point, the number of revertants per plate was scored. The individual results and the mean number of revertants, with the corresponding standard deviation and ratio (mutants obtained in the presence of the test material/mutants obtained in the presence of the vehicle), are recorded.

OTHER EXAMINATIONS:
- The sterility of the S9 mix was checked during each test, before the beginning and at the end of the experiment.

PRELIMINARY TOXICITY TEST:
To assess the toxicity of the test material to the bacteria, six doses (one plate/dose) were tested in the TA 98, TA 100 and TA 102 strains, with or without S9 mix.
The sterility of the test material was also checked during this test.
The test material was freely soluble in the vehicle (distilled water) at 50 mg/mL. Consequently, with a maximum dose volume of 100 µL/plate, the doses were: 10, 100, 500, 1000, 2500 and 5000 µg/plate.
The results were used to determine the dose range for the main test.

Evaluation criteria:

ACCEPTANCE CRITERIA
A study is considered to be valid if the following criteria are met:
- The number of revertants of the controls is within the range of the testing facility’s historical data;
- The number of revertants of the positive controls is higher than that of the controls and within the range of testing facility’s historical data.

EVALUATION CRITERIA
Biological relevance of the results is considered first. In addition, either of the following criteria may be used as an aid for determining a positive response:
- A dose-related increase in the number of revertants, and/or
- A reproducible increase in the number of revertants (i.e. a doubling in at least one strain when compared to that of the controls) for at least one of the doses.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST
No toxicity was noted towards the three strains used, with or without S9 mix.

MUTAGENICITY TESTS
The number of revertants of the vehicle and positive controls was as specified in the acceptance criteria and within the range of the testing facility’s historical data.
The test material did not induce any significant increase in the number of revertants, with or without S9 mix, in any of the five strains.

STERILITY CHECKS
- Test material: The sterility of the test material was found to be satisfactory in the preliminary test.
- S9 mix: The sterility of the S9 mix was found to be satisfactory in the main tests.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: First and Second Mutagenicity Tests without Metabolic Activation

Strain	Dose (µg/plate)	Observations	Mean No. of Revertants per Plate ± SD (ratio)
			First Test	Second Test
TA 1535	0	-	19 ± 6 (-)	15 ± 2 (-)
	30	-	12 ± 3 (0.7)	17 ± 1 (1.1)
	100	-	16 ± 3 (0.8)	13 ± 5 (0.9)
	300	Slight yellow coloured agar	15 ± 6 (0.8)	15 ± 4 (1.0)
	1000	Slight yellow coloured agar	18 ± 1 (1.0)	15 ± 2 (1.0)
	3000	Moderate yellow coloured agar	17 ± 1 (0.9)	16 ± 6 (1.1)
	NaN3 (1)	-	502 ± 25 (26.9)	970 ± 108 (64.7)
TA 1537	0	-	9 ± 5 (-)	9 ± 3 (-)
	30	-	8 ± 4 (0.8)	8 ± 2 (1.0)
	100	-	8 ± 4 (0.9)	8 ± 1 (0.9)
	300	Slight yellow coloured agar	9 ± 4 (1.0)	8 ± 2 (1.0)
	1000	Slight yellow coloured agar	6 ± 5 (0.7)	9 ± 1 (1.0)
	3000	Moderate yellow coloured agar	7 ± 2 (0.7)	7 ± 2 (0.8)
	9AA (50)	-	300 ± 40 (32.2)	1879 ± 89 (216.8)
TA 98	0	-	19 ± 1 (-)	23 ± 2 (-)
	30	-	25 ± 5 (1.3)	21 ± 3 (0.9)
	100	-	15 ± 6 (0.8)	22 ± 2 (1.0)
	300	Slight yellow coloured agar	12 ± 5 (0.6)	16 ± 2 (0.7)
	1000	Slight yellow coloured agar	16 ± 5 (0.8)	15 ± 1 (0.7)
	3000	Moderate yellow coloured agar	13 ± 5 (0.7)	14 ± 4 (0.6)
	2NF (0.5)	-	142 ± 8 (7.3)	157 ± 45 (6.9)
TA 100	0	-	95 ± 10 (-)	75 ± 5 (-)
	30	-	90 ± 9 (1.0)	70 ± 2 (0.9)
	100	-	91 ± 8 (1.0)	72 ± 9 (1.0)
	300	Slight yellow coloured agar	91 ± 14 (1.0)	67 ± 3 (0.9)
	1000	Slight yellow coloured agar	89 ± 11 (0.9)	78 ± 8 (1.0)
	3000	Moderate yellow coloured agar	66 ± 6 (0.7)	60 ± 11 (0.8)
	NaN3 (1)	-	541 ± 133 (5.7)	740 ± 160 (9.8)
TA 102	0	-	259 ± 16 (-)	269 ± 23 (-)
	30	-	246 ± 29 (0.9)	286 ± 18 (1.1)
	100	-	266 ± 7 (1.0)	319 ± 17 (1.2)
	300	Slight yellow coloured agar	256 ± 40 (1.0)	250 ± 36 (0.9)
	1000	Slight yellow coloured agar	273 ± 9 (1.1)	300 ± 26 (1.1)
	3000	Moderate yellow coloured agar	240 ± 56 (0.9)	301 ± 12 (1.1)
	MMC (0.5)	-	2528 ± 458 (9.7)	1641 ± 53 (6.1)

Ratio = number of revertants in the presence of the test material / number of revertants obtained in the presence of the vehicle.

Positive controls: NaN3 = sodium azide; 9AA = 9 -Aminoacridine; 2NF = 2 -Nitrofluorene; MMC = Mitomycin C

No toxicity or precipitation were recorded for any of the plates.

Observations were identical for both the first and second experiments.

Table 2: First and Second Mutagenicity Tests with Metabolic Activation

Strain	Dose (µg/plate)	Observations	Mean No. of Revertants per Plate ± SD (ratio)
			First Test	Second Test
TA 1535	0	-	17 ± 6 (-)	19 ± 4 (-)
	30	-	14 ± 1 (0.9)	12 ± 3 (0.6)
	100	-	13 ± 3 (0.8)	19 ± 4 (1.0)
	300	-	12 ± 3 (0.7)	17 ± 3 (0.9)
	1000	-	14 ± 2 (0.9)	18 ± 4 (0.9)
	3000	Slight yellow coloured agar	9 ± 2 (0.6)	19 ± 7 (1.0)
	2AM (2)	-	229 ± 45 (13.8)	964 ± 88 (50.8)
TA 1537	0	-	15 ± 4 (-)	12 ± 2 (-)
	30	-	11 ± 2 (0.7)	19 ± 4 (1.6)
	100	-	13 ± 2 (0.9)	15 ± 4 (1.3)
	300	-	9 ± 5 (0.6)	12 ± 5 (1.1)
	1000	-	8 ± 4 (0.5)	11 ± 4 (0.9)
	3000	Slight yellow coloured agar	6 ± 2 (0.4)	15 ± 3 (1.3)
	2AM (2)	-	246 ± 47 (16.4)	94 ± 6 (8.1)
TA 98	0	-	25 ± 4 (-)	29 ± 9 (-)
	30	-	16 ± 10 (0.6)	28 ± 7 (1.0)
	100	-	18 ± 10 (0.7)	22 ± 1 (0.8)
	300	-	14 ± 10 (0.6)	25 ± 7 (0.9)
	1000	-	11 ± 6 (0.4)	29 ± 6 (1.0)
	3000	Slight yellow coloured agar	6 ± 1 (0.3)	14 ± 7 (0.5)
	CR (25)	-	270 ± 23 (10.6)	157 ± 20 (5.4)
TA 100	0	-	99 ± 4 (-)	117 ± 17 (-)
	30	-	91 ± 7 (0.9)	106 ± 17 (0.9)
	100	-	135 ± 8 (1.4)	85 ± 17 (0.7)
	300	-	91 ± 22 (0.9)	85 ± 17 (0.7)
	1000	-	100 ± 37 (1.0)	103 ± 6 (0.9)
	3000	Slight yellow coloured agar	82 ± 22 (0.8)	79 ± 12 (0.7)
	2AM (2)	-	1552 ± 209 (15.7)	949 ± 113 (8.1)
TA 102	0	-	305 ± 7 (-)	391 ± 34 (-)
	30	-	290 ± 14 (1.0)	406 ± 49 (1.0)
	100	-	301 ± 36 (1.0)	364 ± 49 (0.9)
	300	-	291 ± 12 (1.0)	394 ± 30 (1.0)
	1000	-	257 ± 12 (0.8)	389 ± 49 (1.0)
	3000	Slight yellow coloured agar	292 ± 6 (1.0)	404 ± 49 (1.0)
	2AM (10)	-	2204 ± 341 (7.2)	879 ± 81 (2.2)

Ratio = number of revertants in the presence of the test material / number of revertants obtained in the presence of the vehicle.

Positive controls: 2AM = 2-Anthramine; CR = Congo Red

No toxicity or precipitation were recorded for any of the plates.

Observations were identical for both the first and second experiments.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

Under the conditions of the test, the test material did not induce mutagenic activity in this bacterial reverse mutation assay on Salmonella typhimurium with or without metabolic activation.

Executive summary:

The potential genotoxicity of the test material was determined in a bacterial reverse mutation assay conducted using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The study was conducted under GLP conditions and in accordance with the standardised guidelines OECD 417 and EU Method B.14.

To assess the toxicity of the test material to the bacteria and define the concentration range for the main tests, a preliminary test was performed using Salmonella strains TA 98, TA 100 and TA 102. These were exposed to the test material at concentrations of 10, 100, 500, 1000, 2500 and 5000 µg/plate (one plate/dose), with and without S9 mix. Based on the observed strong yellow colouration in petri dishes at concentrations from 500 to 5000 µg/plate, the dose levels for the main test were set at 30, 100, 300, 1000 and 3000 µg/plate. The vehicle was distilled water.

In two independent tests, five doses of the test material (three plates/dose level) were tested on each strain, with and without S9 mix. During each test, vehicle and positive controls were run in triplicate. The preincubation method was used (30 minutes at 30 °C) and after 48 to 72 hours of incubation at 37 °C, revertants were scored with an automatic counter.

The test material did not induce any significant increase in the number of revertants, with or without S9 mix, in any of the strains.

Therefore under the conditions of the test, the test material is considered to be non-mutagenic.