Sodium 4-(4-methyl-3-nitrobenzoylamino)benzenesulphonate

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:

2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 4-(4-methyl-3-nitrobenzoylamino)benzenesulphonate

EC Number:

281-719-9

EC Name:

Sodium 4-(4-methyl-3-nitrobenzoylamino)benzenesulphonate

Cas Number:

84029-45-8

Molecular formula:

C14H12N2O6S.Na

IUPAC Name:

sodium 4-(4-methyl-3-nitrobenzoylamino)benzenesulphonate

Details on test material:

- Name of test material (as cited in study report): Nitrotolylsulfanilsäure, Natriumsalz TF
- Physical state: solid/white
- Analytical purity: 91.0 % (w/w)
- Stability under test conditions: > 72 h in DMSO at room temperature
- Storage condition of test material: At room temperature

Method

Species / strain

Species / strain / cell type:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: clearly dissolved, data for the stability in solvent

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None
- Water solubility: soluble in water (50 mg/mL)
- Precipitation: No precipitation was observed
- Other confounding effects:
COMPARISON WITH HISTORICAL CONTROL DATA: performed
ADDITIONAL INFORMATION ON CYTOTOXICITY: No cytotoxic effects up to the highest concentration

Remarks on result:

other: other: reverse mutation assay

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Study Name: 1250301	Study Code: Harlan-CCR 1250301
Experiment: 1250301 VV Pre	Date Plated: 09/06/2009
Assay Conditions:	Date Counted: 12/06/2009

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO			11 ± 2	11 ± 3	22 ± 4	114 ± 8	34 ± 4
	Untreated			12 ± 3	8 ± 3	28 ± 2	142 ± 16	37 ± 4
	Nitrotolylsulfanilsäure,	3 µg		11 ± 3	10 ± 3	24 ± 5	115 ± 11	38 ± 3
	Natriumsalz TF	10 µg		12 ± 3	11 ± 3	23 ± 5	120 ± 6	42 ± 6
		33 µg		9 ± 2	11 ± 1	23 ± 6	130 ± 5	38 ± 4
		100 µg		10 ± 2	12 ± 3	25 ± 5	136 ± 11	42 ± 8
		333 µg		10 ± 2	11 ± 5	22 ± 1	152 ± 2	42 ± 8
		1000 µg		9 ± 1	11 ± 4	20 ± 4	248 ± 12	46 ± 5
		2500 µg		9 ± 3	22 ± 2	32 ± 8	409 ± 20	47 ± 5
		5000 µg		5 ± 0	38 ± 4	40 ± 9	779 ± 5	44 ± 9
	NaN3	10 µg		1737 ± 124			1805 ± 26
	4-NOPD	10 µg				366 ± 6
	4-NOPD	50 µg			88 ± 20
	MMS	3.0 µL						844 ± 34
With Activation	DMSO			12 ± 4	11 ± 5	24 ± 3	132 ± 10	48 ± 9
	Untreated			10 ± 2	9 ± 1	27 ± 1	129 ± 24	60 ± 7
	Nitrotolylsulfanilsäure,	3 µg		14 ± 2	11 ± 4	29 ± 4	137 ± 10	49 ± 5
	Natriumsalz TF	10 µg		9 ± 1	10 ± 2	26 ± 7	122 ± 16	50 ± 7
		33 µg		13 ± 1	11 ± 5	29 ± 4	121 ± 9	47 ± 3
		100 µg		13 ± 2	10 ± 3	27 ± 4	147 ± 12	50 ± 6
		333 µg		12 ± 3	9 ± 1	27 ± 4	171 ± 13	47 ± 4
		1000 µg		12 ± 3	15 ± 1	27 ± 4	281 ± 17	45 ± 7
		2500 µg		11 ± 3	26 ± 5	33 ± 10	458 ± 23	50 ± 3
		5000 µg		19 ± 3	51 ± 4	59 ± 5	735 ± 62	46 ± 4
	2-AA	2.5 µg		216 ± 12	242 ± 19	1753 ± 95	1809 ± 392
	2-AA	10.0 µg						262 ± 15

Key to Positive Controls
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by base pair changes and frameshift in the genome of the strains TA 1537, TA 98 and TA 100.
Therefore, Nitrotolylsulfanilsäure, Natriumsalz TF is considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

The test item Nitrotolylsulfanilsäure, Natriumsalz TF was assessed for its potential to induce gene mutations in the plate incorporation test (experi­ment I) using Salmo­nella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escheri­chia coli strain WP2 uvrA.

The assay was performed with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:           3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal back­ground growth in all strains with and without metabolic activation.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains.

No precipitation of the test item (visible to the unaided eye) was observed in the overlay agar either in the test tubes or on the incubated agar plates.

Substantial and dose dependent increases in revertant colony numbers were observed following treatment with Nitrotolylsulfanilsäure, Natriumsalz TF in strains TA 1537 and TA 100 in the absence of metabolic activation and in strains TA 1537, TA 98, and TA 100 in the presence of metabolic activation (S9 mix). The number of colonies exceeded the threshold of twice the number of the corresponding solvent control in strain TA 98 at 5000 µg/plate in the presence of metabolic activation and in strain TA 100 at 1000 µg/plate and above in the presence and absence of metabolic activation. The number of colonies exceeded the threshold of trice the number of the corresponding solvent control in strain TA 1537 at 5000 µg/plate in the absence and presence of metabolic activation. A slight and dose dependent increase in the number of revertants was also detected in strain TA 98 in the absence of metabolic activation but the threshold of two was not quite reached.

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

In conclusion, it can be stated that during the described mutage­nicity test and under the experimental conditions reported, the test item did induce gene mutations by base pair changes and frame shift mutations in the genome of strains TA 1537, TA 98 and TA 100.