Trisodium bis[(3'-nitro-5'-sulfonato-(6-amino-2-[4-(2-hydroxy-1-naphtylazo)phenylsulfonylamino]pyrimidin-5-azo)benzene-2',4-diolato)]chromate(III)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 04, 1994 - January 14, 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The study was performed only on 4 different strains instead of 5, since followed the OECD 471 of 1983, which was modified later in 1997.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

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:

Trisodium bis[(3'-nitro-5'-sulfonato-(6-amino-2-[4-(2-hydroxy-1-naphtylazo)phenylsulfonylamino]pyrimidin-5-azo)benzene-2',4-diolato)]chromate(III)

EC Number:

418-220-4

EC Name:

Trisodium bis[(3'-nitro-5'-sulfonato-(6-amino-2-[4-(2-hydroxy-1-naphtylazo)phenylsulfonylamino]pyrimidin-5-azo)benzene-2',4-diolato)]chromate(III)

Molecular formula:

C52H32CrN18Na3O20S4

IUPAC Name:

chromium(3+) trisodium bis(6-amino-2-{4-[2-(2-hydroxynaphthalen-1-yl)diazen-1-yl]benzenesulfonamido}-5-[2-(3-nitro-2-oxido-5-sulfonatophenyl)diazen-1-yl]pyrimidin-4-olate)

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Mutations of the bacterial strains used in the study can be described as follows:
Salmonella typhimirium
- TA 1537: his C 3076; rfa-; uvrB- (frame shift mutations)
- TA 98; his D 3052; rfa-; uvrB-;R-factor (frame shift mutations)
- TA 1535: his G 46; rfa-; uvrB- (base-pair substitutions)
- TA 100: his G 46; rfa-; uvrB-;R-factor (base-pair substitutions)
Regular checking of the properties of the Salmonella strains with regard to membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed in the testing laboratory according to Ames test. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.

Metabolic activation:

with and without

Metabolic activation system:

S9 hamster liver microsomal fraction

Test concentrations with justification for top dose:

33.3, 100.0, 333.3, 1000.0, 2500.0, 5000.0 µg/plate. Concentrations covered two logarithmic decades. The choice of the top dose was based on a Range-Finding test.

Vehicle / solvent:

- Solvent: DMF
- Justification for choice of solvent: chosen to its solubility properties and its relative nontoxicity for bacteria.

Controls

Untreated negative controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see Remarks

Remarks:

Sodium azide and 4-NOPD were used for "without metabilic activation" controls, while Congo Red and 2-AA for "with metabolic activation" controls.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation).

NUMBER OF REPLICATIONS
The experiment was conducted in two independent assays. For each strain an dose level, including the controls three plates were used as a minimum.

STORAGE
Strain were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.

PRECULTURES
From the thawed ampoules of the strains 0.5 ml suspension was transferred to 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. This nutrient medium contains per litre:
- 8 g Merck Nutrient Broth
- 5 g NaCl
The bacterial culture was incubated in a shaking water bath for 10 hours at 37 °C.

SELECTIVE AGAR
2.0 % Vogel-Bonner-Glucose-Minimal-Agar was used as selective agar. Each petri was filled with 20 ml of this nutrient medium.
The overlay agar contains per litre:
- 6.0 g Merck Agar Agar*
- 6.0 g NaCl*
- 10.5 mg L-histidine x HCl x H2O*
- 12,2 mg biotin*
* (MERCK, D-64 293 Darmstadt)
Sterilizations were performed at 121° C in an autoclave.

Statistics:

No appropriate statistical method is available since no evaluated statistical procedure can be recommended for analysis of data from the bacterial assay at this time.

Results and discussion

Test results

Species / strain:

other: TA 98, TA 100, TA 1535 and TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES
To evaluate the toxicity of test item, a pre-study was performed with straings TA 98 and TA 100. The plates with the test article showed normal background growth up to 5000.0 ug/plate in both the tested strains.

MAIN ASSSAYS
Toxic effects, evidenced by a reduction in the number of revertants, occurred in strain TA 1535 at 2500.0 µg/plate (without S9 mix) and in strain TA 98 at 1000.0; 2500.0 and 5000.0 µg/plate (with and without S9 mix) in experiment I.
In experiment II toxic effects occurred at 2500.0 and 5000.0 (without S9 mix) and at 1000.0; 2500.0 and 5000.0 µg/plate (with S9 mix) in the strains TA 1535, TA 1537, and TA 98. The background growth of the bacteria (as an additional indication of toxicity) was not influenced. The plates incubated with the test article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all strains used.

No substantial and dose-dependent increases in revertant colony numbers of any of the four tester strains were observed following treatment with test article at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in experiment I. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance in experiment II with and without S9 mix in all strains used.

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

Any other information on results incl. tables

Revertants/plate mean from three plates. Without S9 mix.

Concentration µg/plate	TA 1535		TA 1537		TA 98		TA 100
Experiment	I	II	I	II	I	II	I	II
Neg. control	20	17	13	7	40	36	191	143
Solv. control	13	7	14	6	21	13	108	168
33.3	14	8	8	8	21	17	114	176
100.0	16	6	10	8	19	12	111	168
333.3	13	11	9	8	20	14	134	164
1000.0	10	6	10	8	11	9	101	152
2500.0	7	3	8	2	4	7	81	140
5000.0	8	2	8	1	0	4	89	164
Positive controls
Sodium azide (10 µg/plate)	850	793					734	708
4-NOPD (10 µg/plate)			78	66	453	404

Revertants/plate mean from three plates. With S9 mix.

Concentration µg/plate	TA 1535		TA 1537		TA 98		TA 100
Experiment	I	II	I	II	I	II	I	II
Neg. control	15	12	22	15	35	45	156	127
Solv. control	15	15	11	17	39	53	165	140
33.3	21	15	9	18	35	56	182	144
100.0	21	17	20	18	38	50	163	128
333.3	17	17	19	11	31	43	198	106
1000.0	12	7	13	7	24	20	171	155
2500.0	10	5	9	2	10	5	226	130
5000.0	8	2	8	0	5	0	224	101
Positive controls
2-AA (2.5 µg/plate)	251	216	365	400			1530	1297
Congo Red (500 µg/plate)					263	259

Applicant's summary and conclusion

Conclusions:

The test substance did not induce gene mutations by base pair changes or frameshifts, both with and without metabolic activation.

Executive summary:

The test substance was tested for detecting its potential gene mutagenic activity using the Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537. It should be noted that the study was performed only on 4 different strains of microorganisms instead of 5. This difference is due to the fact that the study was performed according to OECD 471 (1983) which was modified later in 1997.The tests were performed with and without metabolic activation. The test item was examined in two independent assays at 6 concentrations from 3.33 to 5000 µg/plate.

In the experiments performed, no relevant increase of the revertant colony numbers was observed in any Salmonella typhimurium strain tested, in the presence and in the absence of S9 mix. The test substance did not induced point mutations by base pair changes and frameshifts in the genome of the strains of Salmonella typhimurium used.