Disodium 4,4'-bis[6-anilino-[4-[bis(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate

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:

From November 3 to 9, 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted according to internationally accepted testing guidelines and performed according to GLP. The OECD recommended combination of strains was tested.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

Deviations:

yes

Remarks:

(no detrimental impact on the outcome of the study).

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

(no detrimental impact on the outcome of the study).

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 4,4'-bis[6-anilino-[4-[bis(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate

EC Number:

224-073-5

EC Name:

Disodium 4,4'-bis[6-anilino-[4-[bis(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate

Cas Number:

4193-55-9

Molecular formula:

C40H44N12O10S2.2Na

IUPAC Name:

disodium 2-[(1E)-2-[4-({4-[bis(2-hydroxyethyl)amino]-6-(phenylamino)-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]ethenyl]-5-({4-[bis(2-hydroxyethyl)amino]-6-[(cyclohexa-1,3-dien-1-yl)amino]-1,3,5-triazin-2-yl}amino)benzene-1-sulfonate

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

TA 1537, genotype his C 3076; rfa-; uvrB; frame shift mutations.
TA 98, genotype his D 3052; rfa-; uvrB; R-factor; frame shift mutations.
TA 1535, genotype his G 46; rfa-; uvrB; base-pair mutations.
TA 100, genotype his G 46; rfa-; uvrB; R-factor; base-pair mutations.
WP2, genotype trp; trp; uvrA; base-pair substitutions and others

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Experiment II: 33, 100, 333, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

On the day of the experiment, the test article was dissolved in deionised water.
The solvent was chosen because of its solubility properties and its relative non- toxicity to the bacteria.
No precipitation of the test article occurred up to the highest investigated dose.

Controls

Untreated negative controls:

yes

Remarks:

current untreated

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine and 2-aminoanthracene

Remarks:

Without met. act: NaN3 for TA 1535, TA 100; 4-NOPD for TA 1537, TA 98; MMS for WP2 uvrA. With met. act: 2-M for TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA.

Details on test system and experimental conditions:

PRECULTURE
From the thawed ampoules of the strains 0.5 ml suspension was transferred into 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. A solution of 20 µl ampicillin (25 µg/ml) was added to the strains TA 98 and TA 100.
This nutrient medium contains per litre:
8 9 Merck Nutrient Broth (MERCK, 0-64293 Darmstadt)
5 9 NaCI (MERCK, 0-64293 Darmstadt)
The bacterial cultures were incubated in a shaking water bath for 4 hours at 37° C.

Selective Agar: the plates with the selective agar were obtained from E. Merck, 0-64293 Darmstadt.

Overlay Agar: the overlay agar contains per litre:
for Salmonella strains:
6.0 g MERCK Agar Agar*
6.0 g NaCI*
10.5 mg L-Histidine x HCl x H2O*
12.2 mg Biotin*
For Escherichia coli:
6.0 g MERCK Agar Agar*
6.0 g NaCI*
2.5 mg Tryptophan*
* (MERCK, 0-64293 Darmstadt)
Sterilisations were performed at 121°C in an autoclave.

S9 PREPARATION
The bacteria used in this assay do not possess the enzyme systems which, in mammals, are known to convert promutagens into active DNA damaging metabolites. In order to over- come this major drawback an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture.
The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male rats, strain Wistar Hanlbm which received daily applications of 80 mg/kg b.w. Phenobarbital ip. dissolved in aqua deionised and β-Naphthotlavone orally dissolved in corn oil on three subsequent days. The livers were prepared 24 hours after the last treatment.
After decapitation of the anaesthetised animals the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate was diluted 1+3 in KCl and centrifuged cold at 9,000 g for 10 minutes at 4 °C. A stock of the supernatant containing the microsomes was frozen in ampoules and stored at -80 °C. Small numbers of the ampoules are kept at - 20 °C for up to one week before use. The protein content was determined using an analysis kit of BioRad Laboratories, D-80939 Munchen.
The protein concentration in the S9 preparation was 23.7 mg/ml.

Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15% v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCI
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.

PRE-EXPERIMENT TOXICITY
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as described below for the experiment I (plate incorporation test).
Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

DOSE SELECTION
Based upon the results of this pre-experiment the concentrations applied in the main experiments were chosen. The concentration range covered two logarithmic decades. Two independent experiments were performed.
According to the dose selection criteria the test article was tested at the following concentrations: 33, 100, 333, 1000, 2500 and 5000 µg/plate .

TEST PERFORMANCE
For each strain and dose level including the controls, three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µl test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
500 µl S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation)
100 µl Bacteria suspension (cf. test system, pre-culture of the strains)
2000 µl Overlay agar
In the pre-incubation assay 100 µl test solution, 500 µl S9 mix / S9 mix substitution buffer and 100 µl bacterial suspension were mixed in a test tube and shaken at 37 °C for 60 minutes. After preincubation 2.0 ml overlay agar (45 °C) was added to each tube. The mixture was poured on selective agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

Evaluation criteria:

A test article is considered positive if either a dose related and reproducible increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is induced.
A test article producing neither a reproducible and dose related increase in the number of revertants, nor a biologically relevant and reproducibly positive response at any one of the test points is considered non-mutagenic in this system.

A mutagenic response is described as follows:
A test article is considered mutagenic if in the strains TA 98, TA 100, and WP2 uvrA the number of reversions will be at least twice as high and in the strains TA 1535 and TA 1537 at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent and reproducible 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.

Results and discussion

Test results

Test results 1

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:

valid

Positive controls validity:

valid

Test results 2

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:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 100

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

Test results 4

Species / strain:

E. coli WP2

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

Test results 5

Species / strain:

S. typhimurium TA 1535

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:

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of results

without S9 mix

Conc. µg/plate	TA 1535		TA 1537		TA 98		TA 100		WP2 uvr A
	I	II	I	II	I	II	I	II	I	II
Negative control	16	12	20	9	22	15	81	110	40	34
Solvent control	14	13	16	10	19	16	99	131	47	38
Positive control*	1023	951	108	138	283	169	286	453	683	356
33	9	11	18	11	25	20	91	117	56	46
100	17	12	15	10	21	14	55	105	38	28
333	12	13	17	12	20	16	87	112	42	37
1000	14	18	14	11	19	14	46	127	40	35
2500	12	16	17	6	19	20	59	124	53	32
5000	18	18	20	10	19	15	94	127	48	31

with S9 mix

Conc. µg/plate	TA 1535		TA 1537		TA 98		TA 100		WP2 uvr A
	I	II	I	II	I	II	I	II	I	II
Negative control	17	17	17	6	34	27	78	125	70	40
Solvent control	21	15	27	8	28	26	84	127	54	42
Positive control**	120	89	127	93	204	60	416	401	167	112
33	21	20	23	14	25	18	68	119	52	33
100	24	18	16	8	27	24	69	115	44	36
333	17	20	25	12	28	25	77	129	52	44
1000	19	15	22	13	20	20	78	115	50	38
2500	20	17	19	11	38	28	77	115	59	55
5000	19	19	17	9	40	26	81	125	56	49

*Sodium azide (10.0 µg/plate) strains TA 1535 and TA 100

4 -nito-o-phenylene-diamine strains TA 1537 (50 µg/plate) and TA 98 (10.0 µg/plate)

Methyl methane snlfonate (5 µl/plate) strain WP2 uvrA

**2-aminoanthracene (2.5 µg/plate) strains TA 1535, TA 1537, TA 98 and TA 100

2 -aminoanthracene (10.0 µg/plate) strain WP2 11vrA

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

Method

The study was performed to investigate the potential of test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 33, 100, 333, 1000, 2500 and 5000 µg/plate.

Results

The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Conclusion

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifcs in the genome of the strains used. Therefore, test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.