4-amino-N-[4-(aminocarbonyl)phenyl]benzamide

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:

4-amino-N-[4-(aminocarbonyl)phenyl]benzamide

EC Number:

277-874-7

EC Name:

4-amino-N-[4-(aminocarbonyl)phenyl]benzamide

Cas Number:

74441-06-8

Molecular formula:

C14H13N3O2

IUPAC Name:

4-amino-N-(4-carbamoylphenyl)benzamide

Details on test material:

- Name of test material (as cited in study report): p-Aminobenzoylaminobenzamid TF

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

pre-experiment/experiment I: 3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
experiment II: 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: plate incorporation and preincubation test


DURATION
- Preincubation period: 1 hour
- 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:

negative

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

- Precipitation:
Precipitation of the test item was observed in the test tubes from 1000 µg/plate up to 5000 µg/plate in both experiments with and without metabolic activation, and on the incubated agar plates from 333 µg/plate up to 5000 µg/plate in the presence of metabolic activation in both experiments. In the absence of metabolic activation precipitation of the test item was observed on the incubated agar plates from 1000 µg/plate up to 5000 µg/plate in experiment I and from 2500 µg/plate up to 5000 µg/plate in experiment II. The undissolved particles had no influence on the data recording.
- Other confounding effects:
COMPARISON WITH HISTORICAL CONTROL DATA: performed
ADDITIONAL INFORMATION ON CYTOTOXICITY: no toxic effects observed

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of Results Pre-Experiment and Experiment I

Study Name: 1280301	Study Code: Harlan-CCR 1280301
Experiment: 1280301 VV Plate	Date Plated: 28/07/2009
Assay Conditions:	Date Counted: 03/08/2009

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				16 ± 3	10 ± 3	28 ± 5	131 ± 6	67 ± 2
	Untreated				13 ± 5	10 ± 2	31 ± 11	148 ± 10	78 ± 18
	p-Aminobenzoyl		3 µg		12 ± 3	10 ± 1	30 ± 4	128 ± 7	67 ± 7
	aminobenzamid		10 µg		19 ± 2	11 ± 2	30 ± 9	126 ± 17	59 ± 5
	TF		33 µg		18 ± 2	11 ± 2	30 ± 3	140 ± 4	61 ± 10
			100 µg		18 ± 6	12 ± 1	27 ± 3	132 ± 9	61 ± 15
			333 µg		19 ± 2	11 ± 2	23 ± 5	138 ± 4	61 ± 6
			1000 µg		14 ± 4^P	9 ± 3^P	23 ± 5^P	135 ± 9^P	63 ± 2^P
			2500 µg		13 ± 1^P M	7 ± 3^P M	18 ± 2^P M	125 ± 5^P M	56 ± 1^P M
			5000 µg		12 ± 3^P M	6 ± 1^P M	20 ± 3^P M	121 ± 6^P M	48 ± 6^P M
	NaN3		10 µg		1822 ± 157			1971 ± 101
	4-NOPD		10 µg				251 ± 14
	4-NOPD		50 µg			82 ± 24
	MMS		3.0 µL						1074 ± 31
With Activation	DMSO				19 ± 3	16 ± 5	35 ± 7	161 ± 13	66 ± 5
	Untreated				19 ± 4	17 ± 5	39 ± 7	173 ± 10	73 ± 9
	p-Aminobenzoyl		3 µg		18 ± 5	18 ± 3	34 ± 6	146 ± 15	68 ± 11
	aminobenzamid		10 µg		20 ± 5	17 ± 2	39 ± 7	164 ± 6	71 ± 8
	TF		33 µg		17 ± 4	14 ± 1	44 ± 6	163 ± 21	68 ± 1
			100 µg		19 ± 4	12 ± 0	35 ± 4	161 ± 13	69 ± 1
			333 µg		17 ± 2^P M	12 ± 2^P M	25 ± 3^P M	133 ± 6^P M	59 ± 4^P M
			1000 µg		14 ± 2^P M	11 ± 1^P M	24 ± 4^P M	139 ± 7^P M	59 ± 7^P M
			2500 µg		15 ± 2^P M	11 ± 2^P M	25 ± 3^P M	134 ± 10^P M	61 ± 4^P M
			5000 µg		16 ± 1^P M	11 ± 2^P M	25 ± 2^P M	133 ± 7^P M	58 ± 6^P M
		2-AA	2.5 µg		362 ± 23	304 ± 9	2027 ± 65	2756 ± 64
		2-AA	10.0 µg						335 ± 12

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: 1280301	Study Code: Harlan-CCR 1280301
Experiment: 1280301 HV2 Pre	Date Plated: 12/08/2009
Assay Conditions:	Date Counted: 17/08/2009

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 ± 4	12 ± 2	30 ± 4	118 ± 15	44 ± 7
	Untreated			15 ± 6	9 ± 2	37 ± 10	168 ± 11	59 ± 4
	p-Aminobenzoyl	33 µg		13 ± 4	12 ± 6	25 ± 3	122 ± 12	51 ± 3
	aminobenzamid	100 µg		17 ± 2	12 ± 4	30 ± 4	144 ± 28	35 ± 8
	TF	333 µg		13 ± 3	11 ± 3	33 ± 7	117 ± 15	44 ± 11
		1000 µg		12 ± 5	13 ± 1	26 ± 4	122 ± 13	39 ± 15
		2500 µg		12 ± 4^P	9 ± 1^P	31 ± 6^P	121 ± 12^P	36 ± 3^P
		5000 µg		10 ± 2^P M	7 ± 2^P M	28 ± 3^P M	112 ± 3^P M	39 ± 2^P M
	NaN3	10 µg		1487 ± 86			1316 ± 183
	4-NOPD	10 µg				476 ± 17
	4-NOPD	50 µg			127 ± 20
	MMS	3.0 µL						248 ± 13
With Activation	DMSO			24 ± 3	16 ± 6	39 ± 5	149 ± 10	61 ± 4
	Untreated			22 ± 2	20 ± 1	41 ± 7	191 ± 24	69 ± 13
	p-Aminobenzoyl	33 µg		20 ± 2	15 ± 4	32 ± 4	153 ± 14	54 ± 6
	aminobenzamid	100 µg		17 ± 6	13 ± 3	38 ± 5	150 ± 10	58 ± 2
	TF	333 µg		20 ± 7^P	20 ± 3^P	45 ± 5^P	144 ± 19^P	62 ± 10^P
		1000 µg		17 ± 2^P	18 ± 4^P	44 ± 4^P	130 ± 10^P	54 ± 4^P
		2500 µg		14 ± 4^P M	15 ± 3^P M	33 ± 9^P M	124 ± 6^P M	42 ± 5^P M
		5000 µg		14 ± 2^P M	14 ± 2^P M	27 ± 4^P M	130 ± 7^P M	32 ± 3^P M
	2-AA	2.5 µg		267 ± 24	202 ± 24	1134 ± 60	1947 ± 479
	2-AA	10.0 µg						349 ± 10

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.

Executive summary:

The test item p-Aminobenzoylaminobenzamid TF 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.

The assay was performed in two independent experiments both 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

Experiment II:                                   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.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with p-Aminobenzoylaminobenzamid TF 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.