N-[4-(aminocarbonyl)phenyl]-4-nitrobenzamide

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well performed GLP and OECD guideline study

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:

N-[4-(aminocarbonyl)phenyl]-4-nitrobenzamide

EC Number:

298-791-2

EC Name:

N-[4-(aminocarbonyl)phenyl]-4-nitrobenzamide

Cas Number:

93839-21-5

Molecular formula:

C14H11N3O4

IUPAC Name:

N-(4-carbamoylphenyl)-4-nitrobenzamide

Details on test material:

- Name of test material (as cited in study report): p-Nitrobenzoylaminobenzamid trocken

Method

Species / strain

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100, E. coli WP2 uvrA

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

Experiment I (plate incorporation test): 3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II (pre-incubation test): 3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.

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:
Experiment I: plate incorporation test
Experiment II: pre-incubation test

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: S. typhimurium TA 1535, TA 1537, TA 98, TA 100, E. coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see additional information on results

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Precipitation of the test item was observed in the test tubes at 2500 and 5000 µg/plate in both experiments. Precipitation of the test item was also observed at 2500 and 5000 µg/plate with S9 mix in experiment I, and from 1000 - 5000 µg/plate without S9 mix in experiment I and II, and with S9 mix in experiment II.
ADDITIONAL INFORMATION ON CYTOTOXICITY
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.
Minor toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed without metabolic activation in strain TA 1535 at 2500 µg/plate in experiment I, and in strain TA 1537 at 5000 µg/plate without metabolic activation in experiment I and II.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary Tables

   Summary of Results Pre-Experiment and Experiment I

Study Name: 1261901	Study Code: Harlan-CCR 1261901
Experiment: 1261901 VV Plate	Date Plated: 21/04/2009
Assay Conditions:	Date Counted: 24/04/2009

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		21 ± 2	14 ± 2	31 ± 8	182 ± 11	56 ± 2
	Untreated		14 ± 2	13 ± 1	36 ± 3	168 ± 21	50 ± 13
	p-Nitrobenzoylaminobenzamid	3 µg	21 ± 5	10 ± 3	35 ± 2	150 ± 14	52 ± 6
	trocken	10 µg	12 ± 2	8 ± 2	37 ± 5	156 ± 12	59 ± 7
		33 µg	16 ± 1	8 ± 1	36 ± 8	172 ± 32	59 ± 4
		100 µg	17 ± 2	9 ± 1	38 ± 4	174 ± 9	81 ± 8
		333 µg	18 ± 4	8 ± 1	47 ± 6	161 ± 16	59 ± 10
		1000 µg	10 ± 1^P	8 ± 2^P	34 ± 3^P	178 ± 9^P	58 ± 8^P
		2500 µg	9 ± 2^P M	8 ± 1^P M	30 ± 3^P M	171 ± 23^P M	48 ± 4^P M
		5000 µg	12 ± 2^P M	5 ± 2^P M	33 ± 4^P M	168 ± 15^P M	41 ± 3^P M
	NaN3	10 µg	2272 ± 76			2131 ± 47
	4-NOPD	10 µg			576 ± 110
	4-NOPD	50 µg		239 ± 59
	MMS	3.0 µL					1383 ± 41
With Activation	DMSO		19 ± 3	11 ± 2	46 ± 9	169 ± 11	77 ± 5
	Untreated		12 ± 1	11 ± 2	51 ± 13	179 ± 13	76 ± 5
	p-Nitrobenzoylaminobenzamid	3 µg	14 ± 1	10 ± 3	49 ± 1	142 ± 8	71 ± 18
	trocken	10 µg	11 ± 5	12 ± 7	40 ± 3	154 ± 9	60 ± 6
		33 µg	22 ± 7	11 ± 3	43 ± 4	184 ± 13	57 ± 2
		100 µg	21 ± 7	12 ± 2	40 ± 6	183 ± 14	74 ± 16
		333 µg	14 ± 1	12 ± 3	40 ± 6	161 ± 22	69 ± 4
		1000 µg	18 ± 3	12 ± 3	41 ± 4	188 ± 15	63 ± 4
		2500 µg	16 ± 2^P M	9 ± 2^P M	44 ± 7^P M	169 ± 21^P M	77 ± 12^P M
		5000 µg	15 ± 1^P M	8 ± 1^P M	39 ± 5^P M	167 ± 12^P M	45 ± 11^P M
	2-AA	2.5 µg	288 ± 9	256 ± 10	1632 ± 75	1830 ± 55
	2-AA	10.0 µg					337 ± 13

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	P M	Precipitate Manual count

  Summary of Results Experiment II

Study Name: 1261901	Study Code: Harlan-CCR 1261901
Experiment: 1261901 HV2 pre	Date Plated: 07/05/2009
Assay Conditions:	Date Counted: 13/05/2009

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		14 ± 1	13 ± 3	32 ± 9	126 ± 29	55 ± 5
	Untreated		15 ± 7	10 ± 2	31 ± 9	148 ± 10	61 ± 9
	p-Nitrobenzoylaminobenzamid	3 µg	13 ± 9	11 ± 2	25 ± 4	112 ± 13	58 ± 9
	trocken	10 µg	18 ± 4	19 ± 9	27 ± 3	129 ± 11	59 ± 17
		33 µg	16 ± 3	9 ± 2	28 ± 5	128 ± 12	55 ± 4
		100 µg	14 ± 1	11 ± 3	30 ± 6	129 ± 8	59 ± 4
		333 µg	21 ± 5	13 ± 5	25 ± 4	129 ± 27	60 ± 4
		1000 µg	18 ± 3^P	16 ± 4^P	29 ± 3^P	120 ± 8^P	48 ± 9^P
		2500 µg	18 ± 5^P M	8 ± 3^P M	34 ± 3^P M	118 ± 11^P M	54 ± 5^P M
		5000 µg	22 ± 4^P M	6 ± 1^P M	28 ± 5^P M	108 ± 4^P M	41 ± 2^P M
	NaN3	10 µg	1784 ± 70			1846 ± 166
	4-NOPD	10 µg			423 ± 30
	4-NOPD	50 µg		104 ± 14
	MMS	3.0 µL					276 ± 17
With Activation	DMSO		24 ± 2	14 ± 1	37 ± 3	130 ± 15	68 ± 9
	Untreated		28 ± 14	19 ± 5	44 ± 4	168 ± 13	73 ± 6
	p-Nitrobenzoylaminobenzamid	3 µg	21 ± 7	17 ± 3	44 ± 12	149 ± 16	67 ± 6
	trocken	10 µg	24 ± 5	13 ± 1	42 ± 12	123 ± 6	69 ± 16
		33 µg	26 ± 2	15 ± 2	39 ± 12	122 ± 4	71 ± 18
		100 µg	26 ± 4	23 ± 2	42 ± 4	149 ± 6	76 ± 8
		333 µg	11 ± 4	19 ± 8	37 ± 4	138 ± 18	62 ± 4
		1000 µg	26 ± 9^P	17 ± 8^P	43 ± 5^P	133 ± 5^P	71 ± 28^P
		2500 µg	17 ± 3^P M	14 ± 5^P M	40 ± 7^P M	132 ± 8^P M	61 ± 10^P M
		5000 µg	23 ± 5^P M	11 ± 1^P M	38 ± 3^P M	129 ± 18^P M	52 ± 10^P M
	2-AA	2.5 µg	243 ± 6	183 ± 19	1033 ± 137	2019 ± 79
	2-AA	10.0 µg					381 ± 16

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	P M	Precipitate Manual count

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, p-Nitrobenzoylaminobenzamid trocken is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

The test item p-Nitrobenzoylaminobenzamid trocken was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA according to OECD guideline 471.

The assay was performed in two independent experiments both with and without metabolic activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

Minor toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed without metabolic activation in strain TA 1535 at 2500 µg/plate in experiment I, and in strain TA 1537 at 5000 µg/plate without metabolic activation in experiment I and II.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with p-Nitrobenzoylaminobenzamid trocken at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

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 not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, p-Nitrobenzoylaminobenzamid trocken is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.