2-methyl-N-(4-sulfamoylphenyl) prop-2-enamide

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:

18 Sep - 1 Oct 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 Jul 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 08 Jun 2000

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

438-940-2

EC Name:

-

Cas Number:

56992-87-1

Molecular formula:

C10 H12 N2 O3 S

IUPAC Name:

2-methyl-N-(4-sulfamoylphenyl) prop-2-enamide

Test material form:

other: powder aggregated into lumps

Details on test material:

- Name of test material (as cited in study report): SPM-N
- Physical state: white lumps with powder
- Lot/batch No.: T-9314
- Expiration date of the lot/batch: 29 Aug 2008
- Storage condition of test material: at room temperature in the dark

Method

Target gene:

his operon (S. typhimurium strains), trp operon (E. coli strains)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9-mix), prepared from the livers of rats treated with phenobarbital (80 mg/kg bw) and beta-naphthoflavone (100 mg/kg bw)

Test concentrations with justification for top dose:

Dose range finding test: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate for TA 100 and E. coli WP2 uvr A (with and without metabolic activation)
Experiment 1: 33, 100 and 333, 1000 and 3300 µg/plate for TA 98, TA 1535 and TA 1537 (with and without metabolic activation)
Experiment 2: 33, 100, 330, 1000, 3330 μg/plate for all strains (with and without metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide, 9-aminoacridine, 2-nitrofluorene, methylmethanesulfonate, 4-nitroquinoline-N-oxide, 2-aminoanthracene (see 'Any other information on materials and methods incl. tables' for details)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction of bacterial background lawn, increase in size of microcolonies, reduction of revertant colonies

Evaluation criteria:

The assays were considered acceptable if they met the following criteria:
a) the negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain.
b) the positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least 3 times the concurrent vehicle control group.
c) the selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.

A test substance is considered negative (not mutagenic) in the test if:
a) the total number of revertants in tester strain TA 100 is not greater than 2 times the concurrent control, and the total number of revertants in tester strains TA 1535, TA 1537, TA 98 or WP2 uvr A is not greater than 3 times the concurrent control.
b) the negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) the total number of revertants in tester strain TA 100 is greater than 2 times the concurrent control, and the total number of revertants in tester strains TA 1535, TA 1537, TA 98 or WP2 uvr A is greater than 3 times the concurrent control.
b) in case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment.

Statistics:

Mean values and standard deviation were calculated.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance was observed at the start and the end of the incubation period at concentrations of 3330 and 5000 μg/plate (with and without metabolic activation)

RANGE-FINDING/SCREENING STUDIES: A range-finding study was performed with TA 100 and WP2 uvr A, using 8 concentrations from 33-5000 μg/plate (with and without metabolic activation). The highest concentration of the test substance used in the subsequent mutation assay was the level at which the test substance showed limited solubility (3330 µg/plate). The results were presented as part of experiment 1.

COMPARISON WITH HISTORICAL CONTROL DATA: yes; the results fall within the historical data range.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Results of experiment 1

 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates±SD)
		Base-pair substitution type		Frameshift type
		TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537
-S9	Dimethyl sulfoxide	107±10	9±4	23±3	18±3	6±3
-S9	3	108±12	-	24±6	-	-
-S9	10	105±5	-	20±3	-	-
-S9	33	100±13	5±1	27±2	20±8	6±1
-S9	100	104±5	7±2	24±3	22±3	5±1
-S9	333	91±5	7±2	23±4	15±1	6±3
-S9	1000	111±4	9±3	20±4	17±4	12±2
-S9	3300 SP	96±10	13±5	24±4	19±5	13±2
-S9	5000 SP	108±15	-	20±1	-	-
Positive controls, –S9	Name	MMS	SA	4-NQO	2-NF	9-AC
	Concentrations (μg/plate)	650	5	10	10	60
		967±16	987±20	1146±12	963±32	338±16
+S9	Dimethyl sulfoxide	81±11	6±2	19±3	23±9	4±1
+S9	3	89±5	-	22±2	-	-
+S9	10	83±15	-	16±3	-	-
+S9	33	90±16	8±3	19±5	27±5	4±1
+S9	100	76±10	8±5	20±4	21±2	4±2
+S9	333	83±10	8±1	20±2	21±8	7±2
+S9	1000	91±10	8±1	20±3	18±3	6±3
+S9	3330 SP	84±14	9±2	21±4	27±2	9±3
+S9	5000 SP	81±16	-	22±3	-	-
Positive controls, +S9^1	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	1	1	10	1	2.5
		619±72	221±12	330±10	856±5	338±13

MMS = methylmethanesulfonate

SA = sodium azide

4-NQO = 4-nitroquinoline-N-oxide

9-AC = 9-aminoacridine

2-NF = 2-nitrofluorene

2-AA = 2-aminoanthracene

SP = slight precipitate

¹S9-mix contained 5% (v/v) S9-fraction

Table 2: Results of experiment 2

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates±SD)
		Base-pair substitution type		Frameshift type
		TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537
-S9	Dimethyl sulfoxide	107±6	13±6	21±3	20±1	4±1
-S9	33	100±5	11±3	25±8	18±1	4±1
-S9	100	99±23	9±1	21±6	14±2	4±1
-S9	333	95±6	11±2	17±2	22±2	8±4
-S9	1000	128±31	8±3	30±3	21±4	11±6
-S9	3300 SP	93±12	10±3	29±2	16±6	12±5
Positive controls, –S9	Name	MMS	SA	4-NQO	2-NF	9-AC
	Concentrations (μg/plate)	650	5	10	10	60
		976±25	941±25	1026±21	918±83	437±64
+S9	Dimethyl sulfoxide	62±3	7±3	22±9	24±3	3±0
+S9	33	60±4	6±2	25±7	19±3	3±1
+S9	100	61±7	5±2	26±3	27±9	3±1
+S9	333	58±2	4±0	25±7	16±5	4±1
+S9	1000	66±5	6±1	26±4	18±6	7±4
+S9	3330 SP	68±10	4±2	27±6	25±9	10±5
Positive controls, +S9^2	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	2.5	1	10	1	5
		613±26	138±7	294±24	369±36	259±17

MMS = methylmethanesulfonate

SA = sodium azide

4-NQO = 4-nitroquinoline-N-oxide

9-AC = 9-aminoacridine

2-NF = 2-nitrofluorene

2-AA = 2-aminoanthracene

SP = slight precipitate

²S9-mix contained 10% (v/v) S9-fraction

In the second experiment in strain TA 1537, up to 3.0 and 3.3-fold increases in the number of revertant colonies compared to the vehicle control was noted, with and without metabolic activation. As no increase was noted in any other strain in the 2 experiments, the increase was only noted in 1 experiment and the increase was within the historical data range and only noted at precipitating concentrations, this increase is not considered to be biologically relevant.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative