2-[[4-[[4-[(4-amino-9,10-dihydro-9,10-dioxo-3-sulpho-1-anthryl)amino]cyclohexyl]amino]-6-fluoro-1,3,5-triazin-2-yl]amino]benzene-1,4-disulphonic acid, potassium sodium salt

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1977

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Performed in accordance with Ames et al. ‘Ames, B. N.; McCann, J.; Yamasdd, E.: Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mut. Res. 31 (1975) 347-364’.

GLP compliance:

no

Remarks:

Study pre-dates GLP

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

0, 3.15, 10, 31.5, 100, 315, 1000, 3000, 3150 μg/plate

Vehicle / solvent:

The test compounds were stored at 4 °C in the dark and dissolved in dimethylsulfoxide (DMSO) or water on the day of the mutagenicity experiment.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

benzo(a)pyrene

other: 2-Aminoanthracene; Benzo(a)pyrene 4,5-oxide; N-Methyl-N'-nitro-N-nitroguanidine

Details on test system and experimental conditions:

Tissue preparations
Male Sprague Dawley rats (200-300 g) received a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight; Aroclor was diluted with sun flower oil 1:5 v/v) 4-5 days before sacrifice. The liver was homogenized in three volumes of sterile, cold KCI (150 mM, buffered with Na phosphate at pH 7.4). The homogenate was centrifuged at 10,000 g for 10 minutes. One volume of the resulting supernatant was nixed with one volume 24 mM MgCl2-10 mM KCI and one volume 12 mM NADP-15 mM glucose-6-phosphate-150 mM Na phosphate pH 7.4.
This preparation is termed 5-9 Mix. For all experiments fresh tissue preparations were used from animals which were sacrificed on the day of the experiment.

Bacteria
Bacteria were grown over night in nutrient broth (8 g Bacto Nutrient Broth (DIFCO) + 5 g NaCl/liter). For inoculation stock cultures which were stored at -70°C were used.

Mutagenicity experiments
The test compound, 500 μl S-9 Mix (or 500 μ1 150 mM KCI), 100 μl of the bacterial culture, and 2000 μl top agar (0.55 % agar, 0.55 % NaCI, 50 μM histidine, 50 μM biotin, 25 mM Na phosphate pH 7.4 45°C) were mixed in a test tube and poured onto a petridish with minimal agar (1.5 % agar, Vogel-Bonner E medium with 2 % glucose). After incubation for 2-3 days in the dark, colonies (his revertants) were counted.
The person who performed the counting did not know the specifications of the plates.
Every single experiment contained several different positive controls for checking the activity of the metabolizing system and the mutability of the bacteria as well as negative controls in the form of sterility controls and solvent blanks.

Rationale for test conditions:

The mutagenicity experiments were performed as described by Ames with minor modifications.

Statistics:

.

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In a previous investigation mutagenicity for Levafixblau 30170 was observed. This mutagenicity was found only in the presence of S-9 Mix and only at very high doses which seemed to be above the metabolic capacity of the S-9 Mix. Therefore, it was suggested that an impurity rather than Levafixblau itself was responsible for the mutagenicity.
In this study a highly purified preparation from Levofixblau 30170, HRW 2538-57 was tested with Salmonella typhimurium TA 100, TA 1537 and TA 98 in the presence and in the absence of S-9 Mix. In contrast to the originally tested Levafixblau, which was included in this study as a positive control, HRW 2538-57 showed no mutagenic effects with TA 100 in the presence of S-9 Mix. No mutagenicity was observed under all other experimental conditions.

Conclusions:

No mutagenicity was observed under all experimental conditions for HRW 2538-57 (purified Reactive Blue 181).
The substance is not classifiable according to CLP criteria.

Executive summary:

The mutagenicity experiments were performed as described by Ames with minor modifications.

 

In a previous investigation mutagenicity for Levafixblau 30170 was observed . This mutagenicity was found only in the presence of 5-9 Mix and only at very high doses which seemed to be above the metabolic capacity of the S-9 Mix. Therefore, it was suggested that an impurity rather than Levafixblau itself was responsible for the mutagenicity.

 

In this study a highly purified preparation from Levafixblau 30170, HRW 2538-57 was tested with Salmonella typhimurium TA 100, TA 1537 and TA 98 in the presence and in the absence of S-9 Mix. In contrast to the originally tested Levafixblau, which was included in this study as a positive control, HRW 2538-57 showed no mutagenic effects with TA 100 in the presence of S-9 Mix. No mutagenicity was observed under all other experimental conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1978

Report date:

1978

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Performed in accordance with Ames et al. 'Ames, B. N.; McCann, J.; Yamasdd, E.: Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mut. Res. 31 (1975) 347-364’

GLP compliance:

no

Remarks:

Strudy pre-dates GLP

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-[[4-[[4-[(4-amino-9,10-dihydro-9,10-dioxo-3-sulpho-1-anthryl)amino]cyclohexyl]amino]-6-fluoro-1,3,5-triazin-2-yl]amino]benzene-1,4-disulphonic acid, potassium sodium salt

EC Number:

277-179-9

EC Name:

2-[[4-[[4-[(4-amino-9,10-dihydro-9,10-dioxo-3-sulpho-1-anthryl)amino]cyclohexyl]amino]-6-fluoro-1,3,5-triazin-2-yl]amino]benzene-1,4-disulphonic acid, potassium sodium salt

Cas Number:

72987-16-7

Molecular formula:

C29 H26 F N7 O11 S3 . x K . x Na C29H(26-x-y)FK(x)N7Na(y)O11S3

IUPAC Name:

potassium sodium 2-{[4-({4-[(4-amino-9,10-dioxo-3-sulfonato-9,10-dihydroanthracen-1-yl)amino]cyclohexyl}amino)-6-fluoro-1,3,5-triazin-2-yl]amino}benzene-1,4-disulfonate

Test material form:

solid: particulate/powder

Method

Target gene:

histidine

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

up to 2500 μg/plate

Vehicle / solvent:

The test compounds were dissolved in dimethylsulfoxide (DMSO) or water on the day of the mutagenicity experiment.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

Tissue preparations
Male Sprague Dawley rats (200-300 g) received a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight; Aroclor was diluted with sun flower oil 1:5 v/v) 4-5 days before sacrifice. The liver was homogenized in three volumes of sterile, cold KCI (150 mM, buffered with Na phosphate at pH 7.4). The homogenate was centrifuged at 10,000 g for 10 minutes. One volume of the resulting supenantant was mixed with one volume 24 mM MgCl2- 100 mM KCI and one volume 12 mM NADP-15 mM glucose-6-phosphate-150 mM Na phosphate pH 7.4.
This preparation is termed S-9 Mix. For all experiments fresh tissue preparations were used from animals which were sacrificed on the day of the experiment.

Bacteria
Bacteria were grown over night in nutrient broth (8 g Bacto Nutrient Broth (DIFCO) + 5 g NaCl/liter). For inoculation stock cultures which were stored at -70°C were used.

Mutagenicity experiments
The test compound, 500 μl S-9 Mix (or 500 μl 150 mM KCI), 100 μl of the bacterial culture, and 2000 μl top agar (0.55 % agar, 0.55 % NaCI, 50 μM histidine, 50 μM biotin, 25 mM Na phosphate pH 7.4, 45°C) were mixed in a test tube and poured onto a petridish with minimal agar (1.5 % agar, Vogel-Banner E medium with 2 % glucose). After incubation for 2-3 days in the dark, colonies (his+ revertants) were counted.
The person who performed the counting did not know the specifications of the plates.
Every single experiment contained several different positive controls for checking the activity of the metabolizing system and the mutability of the bacteria as well as negative controls in the form of sterility controls and solvent blanks.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

weak mutagenic effect at high doses due to impurity

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

A weak dose dependent mutagenic effect was observed with TA 100 in the presence of S-9 Mix. The compound was not mutagenic in the absence of S-9 Mix. Since in the presence of S-9 Mix the mutagenic effect increased up to the highest dose used (2500 μg), and since this is probably much above the metabolic capacity of the S-9 Mix, it is suggested that an impurity rather than the test substance itself was responsible for the mutagenic effect.
The mutagenic effect was very weak compared to that of the positive controls. No mutagenicity was observed with TA 98, TA 1535 and TA 1537.

Remarks on result:

other: mutagenic effect increased when 1000 μg instead of 315 μg was used. Since this is probably much above the metabolic capacity of the S-9 Mix, it is suggested that an impurity rather than the test substance itself was responsible for the mutagenic effect

Applicant's summary and conclusion

Conclusions:

Reative Blue 181 showed a weak mutagenic effect at high doses in the presence of S-9 Mix with TA 100. Since in the presence of S-9 Mix the mutagenic effect increased up to the highest dose used (2500 μg/plate), and since this is probably much above the metabolic capacity of the S-9 Mix, it is suggested that an impurity rather than he test substance itself was responsible for the mutagenic effect.
Under the other experimental conditions no mutagenic effect was observed with Reactive Blue 181.

Executive summary:

The mutagenicity experiments were performed as described by Ames.

 

eactive Blue 181 was tested for mutagenicity with Salmonella typhimurium TA 100, TA1535, TA 1537 and TA 98 in the presence and in the absence of an activating system (S-9 Mix from induced rat livers). A dose dependent mutagenic effect was observed with TA 100 in the presence of S-9 Mix. The compound was not mutagenic in the absence of S-9 Mix. Since in the presence of S-9 Mix the mutagenic effect increased up to the highest dose used (2500 μg), and since this is probably much above the metabolic capacity of the S-9 Mix, it is suggested that an impurity rather than the test substance itself was responsible for the mutagenic effect.

 

The mutagenic effect was very weak compared to that of the positive controls. No mutagenicity was observed with TA 98, TA 1535 and TA 1537.