2-Naphthalenesulfonic acid, 6-amino-5-[2-[4-[[4-[bis(2-hydroxyethyl)amino]-6-[(2-sulfoethyl)amino]-1,3,5-triazin-2-yl]amino]-2-sulfophenyl]diazenyl]-4-hydroxy-, sodium salt (1:3)

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:

1 August 1995 to 18 August 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, performed 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:

-

EC Number:

419-720-5

EC Name:

-

Cas Number:

182061-89-8

Molecular formula:

C25H26N9O12S3 . 3 Na

IUPAC Name:

trisodium 6-amino-5-{2-[4-({4-[bis(2-hydroxyethyl)amino]-6-[(2-sulfonatoethyl)amino]-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]diazen-1-yl}-4-hydroxynaphthalene-2-sulfonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): M-377
- Physical state: solid
- Appearance: garnet powder
- Storage condition of test material: room temperature and protected from light

Method

Target gene:

Histidine synthesis

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: rfa (all strains), uvr B (all strains), pKM 101 (TA98, TA 100)

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

other: rfa, pKM 101, pAQ1

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Preliminary test:
0, 10, 100, 500, 1000, 2500, 5000 μg/plate (stains; TA 98, TA 100 and TA 102, with and without metabolic activation)

First Mutagenicity test:
0, 125, 250, 500, 1000, 2000 μg/plate (strains; TA 1535, TA 1537, TA 100 and TA 102, with and without metabolic activation)
0, 114.5, 229, 458, 916, 1832 μg/plate (strain; TA 98, with and without metabolic activation)

Second Mutagenicity test:
0, 125, 250, 500, 1000, 2000 μg/plate (stains; TA 1535, TA 1537, TA 98, TA 100 and TA 102; with and without metabolic activation)

Vehicle / solvent:

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

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

congo red

mitomycin C

other: 2-anthramine

Details on test system and experimental conditions:

STUDY DESIGN: A preliminary test, followed by two independent mutagenicity tests.

METHOD OF APPLICATION: preincubation
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.
0.05 to 0.1 mL of the test material solution, 0.5 mL phosphate buffer pH 7.4 (without S9 mix) or 0.5 mL of S9 mix (with S9 mix) and 0.1 mL of the strain were incubated for 30 minutes at 30 °C prior to adding the overlay agar and pouring onto the surface of a minimal agar plate (the agar containing 0.5 % rather than 2 % glucose).

DURATION
- Exposure duration: Plates were incubated inverted at 37 °C for 48 to 72 hours.

NUMBER OF REPLICATIONS: Test concentrations were performed in triplicate

EVALUATION PROCEDURE: Following the total incubation period, revertants were scored with an automatic counter.

OTHER: The sterility of the test solution was checked during the preliminary test. The sterility of the S9 mix was checked during each of the mutagenicity tests at the beginning and end of the experiment.

Evaluation criteria:

The study was considered valid if the following were met:
- The number of revertants of the controls was within the range of historical control data
- The number of revertants of the positive controls was higher than that of the controls and within the range of historical data

Biological relevance of the results was considered first. In addition, the following criteria were used as an aid in the determination of a positive result:
- a dose-related increase in the number of revertants and/or
- a reproducible increase in the number of revertants (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

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 2000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 2000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- Preliminary Toxicity Study
The test material was freely soluble in the vehicle at 50 mg/mL in active material. Consequently, with a maximum dose volume of 100 μL/plate, the doses were 10, 100, 500, 1000, 2500 and 5000 μg/plate expressed in active material.
No precipitation, but slight to strong yellow colouration was observed in the Petri plate when scoring the revertants from 500 to 5000 μg/plate, renders scoring impossible at 2500 and 5000 μg/plate.
No toxicity was noted towards the strains with or without S9 mix.
The sterility of the test material was found to be satisfactory.

- Mutagenicity Study
The number of revertants of the vehicle and positive controls was as specified in the acceptance criteria and were within the range of historical data for the test lab.
The top dose was selected as concentrations higher would interfere with scoring the plates. Slight toxicity was noted towards some of the strains as shown by a decrease in the number of revertants.
The test material did not induce any significant increase in the number of revertants, with or without S9 mix, in any of the tester strains used in the study.
In both mutagenicity tests colouration of the agar was noted from a test concentrations of 144.5 µg/plate increasing in intensity up to the top dose of 2000 µg/plate.
No precipitation was recorded.
No signs of toxicity were recorded.
The sterility of the S9 mix was found to be satisfactory.

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 Mutagenicity Test

Strain	Dose	Without S9		With S9
		Mean revertants/ plate	Ratio	Mean revertants/ plate	Ratio
TA 1535	0	19	-	17	-
	125	14	0.8	13	0.8
	250	15	0.8	17	1.0
	500	16	0.8	17	1.0
	1000	15	0.8	13	0.8
	2000	15	0.8	13	0.8
	NaN3 / 2AM	502	26.9	229	13.8
TA 1537	0	9	-	15	-
	125	7	0.7	6	0.4
	250	6	0.6	8	0.5
	500	5	0.5	8	0.6
	1000	4	0.5	6	0.4
	2000	4	0.5	8	0.5
	9AA / 2AM	300	32.2	246	16.4
TA 98	0	19	-	25	-
	114.5	14	0.7	11	0.4
	229	11	0.6	7	0.3
	458	12	0.6	9	0.4
	916	12	0.6	12	0.5
	1832	14	0.7	6	0.2
	2NF / CR	142	7.3	270	10.6
TA 100	0	95		99
	125	86	0.9	78	0.8
	250	94	1.0	99	1.0
	500	93	1.0	87	0.9
	1000	87	0.9	76	0.8
	2000	82	0.9	75	0.8
	NaN3 / 2AM	541	5.7	1552	15.7
TA 102	0	259	-	305	-
	125	181	0.7	284	0.9
	250	256	1.0	285	0.9
	500	173	0.7	250	0.8
	1000	268	1.0	222	0.7
	2000	186	0.7	217	0.7
	MMC / 2AM	2528	9.7	2204	7.2

Table 2: Second Mutagenicity Test

Strain	Dose	Without S9		With S9
		Mean revertants/ plate	Ratio	Mean revertants/ plate	Ratio
TA 1535	0	15	-	19	-
	125	11	0.8	14	0.7
	250	8	0.6	16	0.9
	500	14	0.9	18	0.9
	1000	11	0.8	13	0.7
	2000	12	0.8	13	0.7
	NaN3 / 2AM	970	64.7	964	50.8
TA 1537	0	9	-	12	-
	125	6	0.7	9	0.7
	250	7	0.8	10	0.9
	500	7	0.8	7	0.6
	1000	9	1.1	5	0.4
	2000	5	0.5	4	0.3
	9AA / 2AM	1879	216.8	94	8.1
TA 98	0	23	-	29	-
	125	18	0.8	18	0.6
	250	14	0.6	16	0.5
	500	15	0.7	18	0.6
	1000	12	0.5	25	0.9
	2000	10	0.5	8	0.3
	2NF / CR	157	6.9	157	5.4
TA 100	0	75	-	117	-
	125	66	0.9	87	0.7
	250	63	0.8	71	0.6
	500	70	0.9	62	0.5
	1000	61	0.8	89	0.8
	2000	51	0.7	76	0.7
	NaN3 / 2AM	740	9.8	949	8.1
TA 102	0	269	-	391	-
	125	243	0.9	433	1.1
	250	275	1.0	293	0.7
	500	288	1.1	141	0.4
	1000	283	1.0	183	0.5
	2000	220	0.8	111	0.3
	MMC / 2AM	1641	6.1	879	2.2

Applicant's summary and conclusion

Conclusions:

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

Under the specific conditions of this assay, the test material gave a negative (i.e. non-mutagenic), response in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA 102 both in the presence and absence of metabolic activation. The study is considered to be reliable, relevant and adequate for risk assessment and classification and labelling purposes.

Executive summary:

The potential of the test material to cause gene mutation in bacterial strains was determined in accordance with standardised guidelines OECD 471 and EU Method B.14. Five strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100 and TA102) were treated in the presence and absence of a metabolic activation system (S9 mix). In two separate assays, the test material did not induce any significant, reproducible increases in the observed number of revertant colonies in any of the tester strains used in the presence or absence of metabolic activation.