disodium 7-{[4-chloro-6-(dodecylamino)-1,3,5-triazin-2-yl]amino}-4-hydroxy-3-[{4-[(4-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl]naphthalene-2-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

Study period:

from July 7 to August 6, 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test conducted according to internationally accepted guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1983)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1992)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Red GS 3848

IUPAC Name:

Red GS 3848

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Method

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:

The test article was tested at the following concentrations in DMF: 33.3; 100.0 ; 333.3; 1000.0; 2500.0; and 5000.0 µg/plate.Positive control substances: 10 µg/plate, without metabolic activation; 2.5 and 500 µg/plate, with metabolic activation.

Vehicle / solvent:

Solvent used: DMFJustification for choice of solvent: solubility properties and relative non toxicity for bacteria.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without S9 mix: NaN3 (TA 1535, TA 100) and 4-NOPD (TA 1537, TA 98); with S9 mix: congo red (TA 98) and 2-AA (TA 1535, TA 1537, TA 100).

Positive control substance:

sodium azide

congo red

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

Details on test system and experimental conditions:

Method of application: preincubation test (experiment I and II).Preincubation test100 µl test solution at each dose level / solvent / negative control / reference mutagen solution (positive control), 500 µl S9 mix / S9 mix substitution buffer, 100 µl bacterial suspension incubated for 10 h at 37 °C. After pre-incubation, addition of 2000 µl overlay agar.Then, the mixture is poured on minimal agar plates. After solidification, plates are incubated upside down for at least 48 h at 37 °C in the dark.Selection agent: selective agar plates.Number of replications: for each strain and dose level, including the controls, 3 plates were used.

Evaluation criteria:

Determination of toxicity in pre-experiment: reduction in the number of spontaneous revertants or clearing of the bacterial background lawn.Accepted conditions for evaluation of results: corresponding background growth on both negative control and test plates; normal range of spontaneous reversion rates.Range of spontaneous reversion frequencies referred to negative control group without metabolic activation (historical control range)TA 1535: 10 - 29TA 1537: 5 - 15TA 98: 15 - 57TA 100: 80 - 183A test substance is considered positive if either a dose related and reproducible increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.A test substance producing neither a dose related and reproducible increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.A significant response is described as follows: a test substance is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strain TA 100 or thrice in strains TA 98, TA 1535 and TA 1537.Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of test substance regardless whether the highest dose induced the criteria described above or not.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

up to 5000 µg/plate.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in all strains at highest tested concentration. in TA 98 strain also at 2500 µg/plate with S9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Pre-experiment on toxicity on TA 98 and TA 100 strainsThe test article concentrations were chosen: 33.3, 100.0, 333.3, 1000, 2500.0 and 5000.0 µg/plate.Main experiments (I and II)Normal background growth up to 5000 µg/plate was noted. Toxic effects appeared as a decrease in revertants rate in all strains: TA 1535 at 5000 µg/plate (exp. I with S9 mix and exp. II without S9 mix); TA 1537 at 5000 µg/plate (exp. II without S9 mix); TA98 at 2500 and 5000 µg/plate (in exp. I with S9 mix and exp. I-II without S9 mix); TA 100 at 5000 µg/plate (in exp. I with and without S9 mix).Weak increase of revertant colony numbers are considered to be not relevant, as this effect was not reproduced in the independent experiment.Historical control data (negative control group without metabolic activation):TA 1535: 10 - 29TA 1537: 5 - 15TA 98: 15 - 57TA 100: 80 - 133

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Mean value of revertants/plate for 3 plates without S9 mix

µg/plate	TA 1535		TA 1537		TA 98		TA 100
	I	II	I	II	I	II	I	II
negative control	16	8	12	8	24	20	117	84
solvent control	12	8	9	5	22	23	95	106
positive control	900	634	98	66	961	527	600	745
33.3	16	7	14	4	18	14	90	115
100.0	13	8	11	8	17	20	79	112
333.3	15	9	8	10	16	21	87	110
1000.0	10	7	7	4	19	16	54	93
2500.0	6	7	6	8	14	14	55	89
5000.0	10	2	8	2	7	10	49	102

Mean value of revertants/plate for 3 plates with S9 mix

µg/plate	TA 1535		TA 1537		TA 98		TA 100
	I	II	I	II	I	II	I	II
negative control	17	19	20	21	37	42	166	146
solvent control	14	14	13	23	30	33	159	161
positive control	254	126	141	151	414	150	1909	1053
33.3	17	11	10	29	22	38	147	184
100.0	14	14	11	18	26	30	157	156
333.3	18	14	15	17	27	26	145	184
1000.0	14	10	13	18	22	19	154	140
2500.0	14	12	12	14	12	19	105	136
5000.0	6	10	8	10	6	17	79	112

 

Normal range of spontaneous reversion rates (without metabolic activation)

historical data	TA 1535		TA 1537		TA 98		TA 100
negative control	10 - 29		5 - 15		15 - 57		80 - 133

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):negativeTest substance did not induce point mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, it is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

Method

Two independent in vitro experiments, according to pre-incubation method using Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, both with and without liver microsomal activation (S9).

The concentration range was determined in a pre-test and the selected concentrations were: 33.3, 100.0, 333.3, 1000.0 2500.0 and 5000 µg/plate. Positive, negative and solvent controls with and without metabolic activation were used.

Results

In the pre-experiment on toxicity on TA 98 and TA 100 strains, the test concentrations were chosen.

In the main experiments (I and II), toxic effects evident as a reduction in the number of revertants occurred in all strains: TA 1535 at 5000 µg/plate (exp. I with S9 mix and exp. II without S9 mix); TA 1537 at 5000 µg/plate (exp. II without S9 mix); TA98 at 2500 and 5000 µg/plate (in exp. I with S9 mix and exp. I-II without S9 mix); TA 100 at 5000 µg/plate (in exp. I with and without S9 mix).

The plates incubated with the test article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all the strains used.

No substantial increase in revertant colony numbers of any of the four tester strains was observed following treatment with the substance at any dose level, neither in presence nor in 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.