A mixture of: N,N-diethylpropane-1,3-diamine 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate; 2,2-iminodiethanol 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate; 2-methylaminoethanol 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted according to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1983)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

A mixture of: N,N-diethylpropane-1,3-diamine 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate; 2,2-iminodiethanol 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate; 2-methylaminoethanol 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate

EC Number:

403-410-1

EC Name:

A mixture of: N,N-diethylpropane-1,3-diamine 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate; 2,2-iminodiethanol 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate; 2-methylaminoethanol 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate

Cas Number:

114565-65-0

Molecular formula:

C18 H16 N8 O3 S2 . x C7 H18 N2 . x C4 H11 N O2 . x C3 H9 N O

IUPAC Name:

(3-aminopropyl)diethylamine; 2-(methylamino)ethan-1-ol; 2-[(2-hydroxyethyl)amino]ethan-1-ol; 6-methyl-2-{4-[2-(triaminopyrimidin-5-yl)diazen-1-yl]phenyl}-1,3-benzothiazole-7-sulfonic acid

Details on test material:

- Name of test material (as cited in study report): FAT 11´184/B
- Physical state: solid
- Analytical purity: 89.6%
- Lot/batch No.: Op. 1 BD 823/8
- Expiration date of the lot/batch: January, 1993
- Stability under test conditions: >8 h in water, ethanol, acetone, DMSO and DMF

Method

Target gene:

All Salmonella Typhimurium strains are histidine auxotrophic mutants.

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: TA 1537: his C 3076, rfa-, uvrB-; TA 98: his D 3052, rfa-, uvrB-, R-factor; TA 1535: his G 46, rfa-, uvrB-; TA 100: his G 46, rfa-, uvrB-, R-factor

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

other: TA 1538: his D 3052, rfa-, uvrB-

Metabolic activation:

with and without

Metabolic activation system:

Mixture of co-factors with S9 fraction of liver from Wistar rats induced with Aroclor 1254.

Test concentrations with justification for top dose:

A preliminary toxicity test was carried out with concentrations of 1, 3.3, 10, 33.3, 100, 333.3, 1000, and 5000 µg/plate with strains TA 98 and TA 100. Since no effects on background growth were seen, the top dose level was chosen as highest concentration in the main experiments:
Experiment I: 10, 33.3, 100, 333.3, 1000, 5000 µg/plate
Experiment II: 10, 100, 333.3, 1000, 3333.3, 5000 µg/plate
Results of the preliminary test were reported as part of the main experiment I.

Vehicle / solvent:

DMSO; The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

concurrent untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below

Controls 2

Positive controls:

yes

Remarks:

without S9-mix

Positive control substance:

sodium azide

Remarks:

TA1535, TA 100

Migrated to IUCLID6: 10 µg/plate

Controls 3

Positive controls:

yes

Remarks:

without S9-mix

Positive control substance:

other: 50 µg/plate 4-nitro-o-phenylene-diamine

Remarks:

TA 1537, TA 1538, TA 98

Controls 4

Positive controls:

yes

Remarks:

with S9-mix

Positive control substance:

other: 10 µg/plate 2-aminoanthracene

Remarks:

TA 1535, TA 1537, TA 1538, TA 98, TA 100

Details on test system and experimental conditions:

METHOD OF APPLICATION (plate incorporation test)
In each experiment 0.1 ml of the test substance or the vehicle, 0.1 ml of a bacterial culture (in nutrient broth), 0.5 ml S9-mix (with metabolic activation) or S9-mix substitution buffer (without metabolic activation) in 2.0 ml of overlay agar were mixed together. In the pre-incubation assay soft agar was added after an incubation period of 1 h at 37 °C. The plates were incubated for 72 hours at 37 °C in darkness. Each concentration and the controls were tested in triplicate.

DETERMINATION OF CYTOTOXICITY
Toxicity was assessed as clearing of the bacterial background lawn and/or as reduction of spontaneous revertants.

Evaluation criteria:

The test article was considered as positive if either a significant dose-response relationship in the increased number of revertants or a significant and reproducible increase for at least one test concentration was induced.
A test article was considered as mutagen if the number of revertants was at least twice as high in strain TA 100 and at least three times as high as the spontaneous reversion rate in strains TA 1535, TA 1537, TA 1538, and TA 98.

A dose-dependent increase in the number of revertants was 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 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

Test results 2

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 3

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

a significant and reproducible dose-dependent increase of revertants was seen.

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:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

a significant and reproducible dose-dependent increase of revertants was seen.

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 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the absence of S9-mix the number of spontaneous revertants was decreased.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

A reduction of spontaneous revertants was seen for strain TA 100 without S9-mix only. No effect on the normal background growth was seen up to 5000 µg/plate with and without S9-mix in any strain.
A significant and reproducible dose-dependent increase in revertant colony numbers was seen in the Salmonella typhimurium strain TA 1538 with and without S9-mix and TA 98 without S9 mix.

Remarks on result:

other: strain/cell type: see above

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Revertants/plate (mean of three plates) in the Ames test with and without metabolic activation

	Revertants/plate, without S9 mix (mean of three plates)
Strain	TA 1535		TA1537		TA 98		TA 100		TA 1538
Experiment	I	II	I	II	I	II	I	II	I	II
Negative control	17	11	10	12	16	29	81	112	14	7
Solvent control	13	15	8	13	18	23	86	94	14	12
Positive control	1054	578	195	173	1856	1328	754	767	2102	1664
10	13	10	12	7	17	27	88	88	14	9
33.3	18	---	9	---	14	---	95	---	17	---
100	13	11	7	9	13	16	84	79	12	13
333.3	15	9	8	11	13	22	90	74	16	13
1000	11	8	10	8	34	30	72	87	32	29
3333.3	---	8	---	8	---	41	---	88	---	50
5000	13	11	9	10	49	66	46	71	89	81
	Revertants/plate, with S9 mix (mean of three plates)
Negative control	10	8	14	10	26	29	79	83	25	17
Solvent control	8	10	9	7	25	32	58	86	18	16
Positive control	296	240	373	354	2024	1920	2229	1548	1840	990
10	10	7	12	6	26	24	65	70	21	20
33.3	10	---	14	---	29	---	57	---	20	---
100	14	8	12	9	36	20	72	88	27	21
333.3	12	12	10	10	32	30	66	95	29	21
1000	10	9	11	8	36	26	72	98	35	28
3333.3	---	6	---	11	---	29	---	100	---	34
5000	15	8	14	12	43	46	72	95	51	47
Experiment I and II: plate incorporation method

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive