Benzamide, N-[[4-[(cyclopropylamino)carbonyl]phenyl]sulfonyl]-2-methoxy-

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:

17 - 27 Nov 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 Jul 1997 (corrected 26 Jun 2020)

Deviations:

yes

Remarks:

The sole indicator of S9 efficacy was 2-aminoanthracene (2-AA). Historical control ranges are present as median values, with a semi Q-range, without 95th or 99th percentile.

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Umwelt und Naturschutz, Landwirtschaft und Verbraucherschutz des Landes Nordrhein-Westfalen, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

485-320-2

EC Name:

-

Cas Number:

221667-31-8

Molecular formula:

C18H18N205S

IUPAC Name:

N-[4-(cyclopropylcarbamoyl)benzenesulfonyl]-2-methoxybenzamide

Method

Target gene:

his operon

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:

Type and composition of metabolic activation system:
Cofactor supplemented post-mitochondrial fraction (S9 mix) was prepared from the livers of at least six adult male Sprague Dawley rats, of approximately 200 to 300 g in weight. For enzyme induction, the animals received a single intraperitoneal injection of Aroclor 1254, dissolved in com oil, at a dose of 500 mg/kg body weight, five days prior to sacrifice.

The protein concentration of the S9 preparation was 37.6 mg/mL in the both experiments. 70 mL of the co-factor contained: 162.6 mg MgCI2 x 6H20, 246 mg KCI, 179.1 mg Glucose-6-phosphate, disodium salt, 315 mg NADP, disodium salt in 100 mM sodium phosphate buffer. S9 fraction was thawed and mixed with S9 cofactor solution and, if needed, 0.15 M KCI, to result in a final concentration of approx. 10% v/v in the S9 mix and approx. 1.8% in the final culture medium.

Prior to first use, each batch was checked for its metabolizing capacity by using reference mutagens; appropriate activity was demonstrated. At the beginning of each experiment four aliquots of the S9 mix were plated (0.5 ml per plate) in order to assess its sterility. This was repeated after completion of test tube plating. The sterility control plates were then incubated for 48 hours at 37°C. No indication of contamination of S9 mix was found.

Test concentrations with justification for top dose:

First experiment: 16, 50, 158, 500, 1581 and 5000 µg/plate with and without metabolic activation
Second experiment: 16, 50, 158, 500, 1581 and 5000 µg/plate with and without metabolic activation

5000 µg/plate was selected as the highest test concentration based on the results of first experiment, which also served as the pre-test for toxicity. No significant cytotoxicity and no precipitation was observed up to and including the highest concentration.

Vehicle / solvent:

- Vehicle/solvent used: DMSO (0.1 mL/plate)
- Justification for choice of solvent/vehicle: Solubility of the test compound

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

cumene hydroperoxide

mitomycin C

other: Nitrofurantoin (NF): TA 100, 0.2 μg/plate in DMSO, -S9; 4-nitro-1,2-phenylene diamine (4-NPDA): TA98 0.5 µg/plate and TA1537 10 µg/plate in DMSO, -S9; 2-aminoanthracene (2-AA), TA 1535, TA 100, TA 1537, TA 98 and TA 102, 3 µg/plate in DMSO, +S9.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar for plate incorporation test (experiment I) and in bacterial suspension for preincubation (experiment II).

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min
- Exposure duration/duration of treatment: 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Reduction in background growth; reduction in mutant count per plate; titer.

Evaluation criteria:

Acceptance of an assay:
1. The negative controls had to be within the expected range, as defined by published data (e.g. Maron and Ames, 1983) and/ or the laboratories' own historical data.
2. The positive controls had to show sufficient effects, as defined by the laboratories' experience.
3. Titer determinations had to demonstrate sufficient bacterial density in the suspension.
Even if the criteria for points 1 and 2 were not met, a trial was accepted if it showed mutagenic activity of the test compound.

Criteria for a positive result:
A reproducible and dose-related increase in mutant counts of at least one strain (For TA1535, TA100 and TA98 about twice that of negative controls, for TA1537 at least threefold; for TA102 an increase of about 100 mutants should be reached). Otherwise the result is evaluated as negative.

Statistics:

Means and standard deviation of triplicate plates were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and 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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and 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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and 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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and 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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and 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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

STUDY RESULTS
- No precipitation of the test item occurred up to the highest investigated dose in both experiments. No indication of a bacteriotoxic effect was observed at doses of up to and including 5000 µg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. No inhibition of growth was noted as well.
- None of the five strains concerned showed in the plate incorporation test a dose related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to the tests with and without S9 mix and was confirmed by the results of the pre-incubation trials.

STABILITY IN VEHICLE:
- The test substance was stable in DMSO at room temperature at concentrations ranging from 0.01 mg/mL to 200 mg/mL for at least 24 hours. An interval which
covers the time range from preparation of the formulation to last treatment.
- Content as % of nominal value after storage time in hours: 0.01 mg/mL, 96% at 0 and 24 hours. 200 mg/mL, 92 and 91%, at 0 and 24 hours, respectively.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The experimental data is well comparable with the provided historical control data (see Attachment 1 for historical control data).
- The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, mitomycin C, cumene hydroperoxide and 2-aminoanthracene increased mutant counts to well over those of the negative controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix.

Any other information on results incl. tables

Table 1: Test results (experiment I, plate incorporation)

Without S9	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		TA 1535	TA 1537	TA 98	TA 100	TA 102
	Solvent control (DMSO)	10 ± 2	7± 3	16 ± 3	126 ± 6	217 ± 22
	16	12 ± 3	6 ± 1	17 ± 6	121 ± 5	191 ± 26
	50	10 ± 5	7 ± 1	16 ± 3	116 ± 19	163 ± 21
	158	15 ± 4	6 ± 2	18 ± 4	115 ± 26	208 ± 24
	500	15 ± 4	7 ± 2	11 ± 2	118 ± 12	175 ± 29
	1581	13 ± 8	7 ± 1	11 ± 4	110 ± 12	147 ± 4
	5000	10 ± 1	6 ± 1	9 ± 2	115 ± 11	154 ± 20
	Positive controls (µg/plate)	Na-azide (10)	4-NPDA (10)	4-NPDA (0.5)	NF (0.2)	MMC (0.2)
	Mean (No. of colonies/plate)	407 ± 41	81 ± 7	140 ± 9	337 ± 14	428 ± 44
With S9	Solvent control (DMSO)	10 ± 4	8 ± 2	21 ± 6	138 ± 18	235 ± 18
	16	9 ± 3	7 ± 1	17 ± 4	117 ± 14	191 ± 43
	50	9 ± 2	8 ± 2	14 ± 5	98 ± 18	189 ± 10
	158	10 ± 5	7 ± 2	22 ± 4	142 ± 16	240 ± 58
	500	9 ± 2	8 ± 2	24 ± 6	131 ± 15	261 ± 14
	1581	11 ± 2	7 ± 1	14 ± 5	123 ± 9	234 ± 47
	5000	7 ± 2	7 ± 1	18 ± 2	122 ± 16	205 ± 31
	Positive controls (µg/plate)	2-AA (3)	2-AA (3)	2-AA (3)	2-AA (3)	2-AA (3)
	Mean (No. of colonies/plate)	105± 12	72 ± 8	556 ± 147	1229 ± 72	471 ± 35

2-AA: 2-Aminoanthracene

MMC: Mitomycin C

4-NPDA: 4-Nitro-1,2-phenyiene diamine

NF: Nitrofurantoin

Na-azide: Sodium azide

Table 2: Test results (experiment II, pre-incubation)

Without S9	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		TA 1535	TA 1537	TA 98	TA 100	TA 102
	Solvent control (DMSO)	26 ± 1	8± 1	14 ± 2	116 ± 4	184 ± 39
	16	21 ± 6	8 ± 1	12 ± 2	114 ± 6	173 ± 9
	50	26 ± 1	8 ± 2	13 ± 2	101 ± 5	158 ± 7
	158	22 ± 6	9 ± 2	15 ± 5	124 ± 11	209 ± 21
	500	27 ± 1	9 ± 2	13 ± 4	115 ± 12	179 ± 14
	1581	23 ± 3	7 ± 0	16 ± 3	130 ± 6	173 ± 4
	5000	15 ± 5	7 ± 2	15 ± 1	118 ± 12	168 ± 6
	Positive controls (µg/plate)	Na-azide (10)	4-NPDA (10)	4-NPDA (0.5)	NF (0.2)	Cumene (50)
	Mean (No. of colonies/plate)	535 ± 35	94 ± 10	135 ± 3	428 ± 12	414 ± 34
With S9	Solvent control (DMSO)	12 ± 4	8 ± 1	20 ± 4	112 ± 3	196 ± 9
	16	14 ± 2	9 ± 2	17 ± 3	98 ± 8	207 ± 32
	50	13 ± 1	7 ± 1	20 ± 5	95 ± 10	190 ± 15
	158	10 ± 3	8 ± 1	22 ± 2	126 ± 21	187 ± 34
	500	14 ± 3	7 ± 2	22 ± 2	105 ± 2	205 ± 18
	1581	10 ± 3	8 ± 3	19 ± 5	96 ± 4	194 ± 62
	5000	13 ± 2	8 ± 2	22 ± 5	118 ± 15	183 ± 24
	Positive controls (µg/plate)	2-AA (3)	2-AA (3)	2-AA (3)	2-AA (3)	2-AA (3)
	Mean (No. of colonies/plate)	156± 18	210 ± 13	741 ± 111	1344 ± 64	405 ± 25

2-AA: 2-Aminoanthracene

Cumene: Cumene hydroperoxide

4-NPDA: 4-Nitro-1,2-phenyiene diamine

NF: Nitrofurantoin

Na-azide: Sodium azide

Applicant's summary and conclusion

Conclusions:

The study was performed according to OECD guideline 471 and compliant with GLP. Under the conditions of the assay, the test item was not mutagenic in S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and in TA 102 with and without metabolic activation.