Reaction mass of (disodium 4-[[4-(acetylmethylamino)-2-sulphonatophenyl]amino]1-amino-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate and sodium sulphate and sodium chloride)

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:

1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study according to GLP regulation; full report available

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

pentasodium 1-amino-4-{[4-(N-methylacetamido)-2-sulfonatophenyl]amino}-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate chloride sulfate

EC Number:

944-710-7

Molecular formula:

not available because multi-constituent substance

IUPAC Name:

pentasodium 1-amino-4-{[4-(N-methylacetamido)-2-sulfonatophenyl]amino}-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate chloride sulfate

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, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

rat Liver S-9

Test concentrations with justification for top dose:

0 - 312,5 - 625 - 1250 - 2500 - 5000 µg/plate

Vehicle / solvent:

water

Controls

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-anthramine (2AM)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation;

DURATION
- Preincubation period: 60 min.
- Exposure duration: 48-72 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth;

Evaluation criteria:

Treatment of results
In each experiment, for each strain and for each experimental point, the number of revertants per plate was scored. The individual results and the mean number of revertants, with the corresponding standard deviation and ratio (mutants obtained in the presence of the test substance/mutants obtained in the presence of the vehicle), are presented in a table.

Acceptance criteria
This study would be considered valid since the following criteria are fully met:
- the number of revertants in the vehicle controls is consistent with the range of our historical data (appendix 2),
- the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with the range of our historical data.

Evaluation criteria
A reproducible two-fold increase in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Water solubility: 100g/L
- Precipitation: No

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant toxicity was noted towards all the strains used, both with and without S9 mix.

Remarks on result:

other: strain/cell type: TA 100

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

First experiment with S9

Strain	Dose (µg/plate)	Revertants / Plate (Mean)	Standard eviation	Ratio
TA 100	0	95	16	1,00
	312,5	92	5	0,97
	625	111	11	1,17
	1250	112	8	1,18
	2500	105	9	1,11
	5000	101	22	1,07
	2AM	2286	94	24,06
TA102	0	343	33	1,00
	312,5	352	13	1,03
	625	351	19	1,02
	1250	348	8	1,02
	2500	359	25	1,05
	5000	338	35	0,99
	2AM	2139	443	6,24
TA 98	0	39	8	1,00
	312,5	47	2	1,21
	625	45	2	1,15
	1250	39	4	0,99
	2500	46	5	1,17
	5000	54	3	1,39
	2AM	737	57	18,9
TA 1535	0	18	6	1,00
	312,5	21	5	1,21
	625	26	2	1,45
	1250	28	10	1,58
	2500	19	3	1,06
	5000	19	7	1,09
	2AM	278	65	15,72
TA 1537	0	19	8	1,00
	312,5	24	4	1,26
	625	19	5	0,98
	1250	15	5	0,79
	2500	24	5	1,22
	5000	21	5	1,07
	2AM	95	11	4,90

 

Second experiment with S9

Strain	Dose (µg/plate)	Revertants / Plate (Mean)	Standard Deviation	Ratio
TA 100	0	94	10	1,00
	312,5	144	9	1,54
	625	178	24	1,90
	1250	200	15	2,13
	2500	271	32	2,90
	5000	168	4	1,79
	2AM	1153	32	12,31
TA102	0	365	27	1,00
	312,5	423	19	1,16
	625	403	5	1,10
	1250	403	16	1,10
	2500	397	9	1,09
	5000	374	10	1,02
	2AM	2670	123	7,31
TA 98	0	39	4	1,00
	312,5	49	8	1,26
	625	54	6	1,38
	1250	57	5	1,45
	2500	56	9	1,44
	5000	44	13	1,13
	2AM	1098	240	28,16
TA 1535	0	20	4	1,00
	500	20	3	0,98
	1000	19	5	0,95
	2000	17	1	0,85
	2500	21	3	1,02
	5000	18	2	0,90
	2AM	214	9	10,54
TA 1537	0	16	2	1,00
	312,5	17	3	1,02
	625	20	8	1,24
	1250	16	1	1,00
	2500	14	6	0,86
	5000	10	2	0,63
	2AM	82	15	5,02

Third Experiment with S9

Strain	Dose (µg/plate)	Revertants / Plate (Mean)	Standard Deviation	Ratio
TA 100	0	146	8	1,00
	625	244	7	1,67
	1250	305	11	2,09
	2500	341	6	2,34
	3000	341	32	2,34
	5000	309	46	2,12
	2AM	1117	144	7,65

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive with metabolic activation only in strain TA 100 after preincubation

The test item showed mutagenic activity in a bacterial reverse mutation test on Salmonella typhimuriumTA 100 strain, with metabolic activation and preincubation method.

Executive summary:

The objective of this study was to evaluate the potential of the test substance to induce reverse mutation in Salmonella typhimurium.

The test item was tested in two independent experiments, with or without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

A third experiment was performed with S9 mix.

The first experiment was performed according to the direct plate incorporation method. The second and the third with S9 mix were performed according to the preincubation method (60 minutes, 37C).

Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used.

Each strain was exposed to five dose-levels of the test substance (three plates/dose-level).

After 48 to 72 hours of incubation at 37C, the revertant colonies were scored.

The test substance was dissolved in distilled water.

Results

The test substance was freely soluble in the vehicle at 54.348 mg/ml, corresponding to 50 mg of the active material/ml (purity of the commercial product: 92%). Since the test substance was freely soluble and non-toxic in the preliminary toxicity test, the highest dose-level was 5000 µg/plate, according to the criteria specified in the guidelines.

The selected treatment-levels for the mutagenicity experiments were: 312.5, 625, 1250, 2500 and 5000 µg/plate, for all strains in the first and second experiments, except for the TA 1535 strain in the second experiment with S9 mix where the treatment-levels were: 500, 1000,2000,2500 and 5000 µg/plate.

In the third experiment, the dose-levels were: 625, 1250, 2500, 3000 and 5000 µg/plate.

Slight to strong blue coloration was observed in the Petri plates when scoring the revertants at dose-levels. This coloration did not impede the automatic counting of the bacterial colonies. No precipitate was observed at any dose-level.

No relevant toxicity was noted towards all the strains used, both with and without S9 mix.

The test substance did not induce any significant increase in the number of revertants, with S9 mix and without S9 mix, in any of the five strains using the direct plate incorporation method.

With S9 mix and the preincubation method, a significant increase in the number of revertants, in comparison to the vehicle control, was noted in the TA 100 strain, at the dose-levels of 1250 and 2500 µg/plate. In addition, a dose-related increase was observed in this strain, at all dose-levels tested, except at the highest dose-level of 5000 µg/plate where a slight toxicity could have masked the increase in the number of revertants.

A third experiment was performed under the same conditions, in order to check the reproducibility of the significant increase observed. In this third experiment, the significant induction of the number of revertants was confirmed.

The number of revertants of the vehicle and positive controls was as specified in the acceptance criteria.

The study was therefore considered as valid.

Conclusion

Under our experimental conditions, the test substance showed mutagenic activity in this bacterial reverse mutation test on Salmonella typhimuriumTA 100 strain, with metabolic activation and preincubation method.