Benzamide, 3-amino-4-methoxy-N-methyl-

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:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-amino-4-methoxy-N-methylbenzamide

EC Number:

813-034-3

Cas Number:

878160-14-6

Molecular formula:

C9H12N2O2

IUPAC Name:

3-amino-4-methoxy-N-methylbenzamide

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Purity: 99.9 % (w/w)

Method

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-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

Vehicle / solvent:

Solvent used: DMSO
Justification for choice of solvent: best suitable solvent, because of its solubility properties and its relative nontoxicity to the bacteria

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

exposure duration: 72 hours

Number of replications: 3 plates (each concentration including controls)

DETERMINATION OF CYTOTOXICITY: Evident as 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, and WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

in strains TA 1537 and TA 98 in the presence of metabolic activation (S9 mix)

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: not soluble
Precipitation: No precipitation of the test item occurred up to the highest investigated dose.
Other confounding effects: none

COMPARISON WIT HISTORICAL CONTROL DATA: performed
ADDITIONAL INFORMATION ON CYTOTOXICITY: 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.

Remarks on result:

other:

Remarks:

, the test item did induce gene mutations by frameshifts in the genome of strain TA 1537 and TA 98

Any other information on results incl. tables

Summary of Experiment I

Date plated: 01 November 2016

Date counted: 04 November 2016

	Test Group	Dose Level per Plate	TA 1535 Revertant Colony Counts ( Mean±SD)	TA 1537  Revertant Colony Counts ( Mean±SD)	TA 98  Revertant Colony Counts ( Mean±SD)	TA 100  Revertant Colony Counts ( Mean±SD)	WP2 uvrA  Revertant Colony Counts ( Mean±SD)
Without	Solvent		20 ± 6	10 ± 1	22 ± 5	167 ± 3	42 ± 11
Activation	Untreated		22 ± 4	8 ± 2	21 ± 2	153 ± 9	46 ± 4
	Test Item	3 µg	18 ± 7	9 ± 2	27 ± 9	159 ± 20	36 ± 5
		10 µg	16 ± 1	8 ± 3	25 ± 4	155 ± 4	34 ± 5
		33 µg	23 ± 7	10 ± 4	30 ± 8	159 ± 9	24 ± 8
		100 µg	22 ± 4	10 ± 3	21 ± 5	162 ± 10	40 ± 6
		333 µg	16 ± 4	10 ± 5	23 ± 3	147 ± 19	34 ± 3
		1000 µg	22 ± 1	11 ± 3	21 ± 2	152 ± 8	139 ± 8
		2500 µg	21 ± 5	8 ± 2	20 ± 7	164 ± 21	28 ± 2
		5000 µg	21 ± 5	7 ± 2	24 ± 8	166 ± 17	27 ± 4
	NaN3	10 µg	1526 ± 89			2477 ± 91
	4 -NOPD	10 µg			554 ± 69
	4 -NOPD	50 µg		85 ± 21
	MMS	2.0 µL					1043 ± 25

With	DMSO		17 ± 3	10 ± 1	36 ± 5	109 ± 11	39 ± 2
Activation	Untreated		11 ± 2	14 ± 5	36 ± 4	139 ± 13	53 ± 8
	Test Item	3µg	14 ± 3	9 ± 2	40 ± 5	95 ± 11	47 ± 6
		10µg	15 ± 1	10 ± 4	39 ± 8	96 ± 12	48 ± 6
		33µg	16 ± 6	10 ± 4	32 ± 8	106 ± 19	45 ± 4
		100µg	11 ± 4	13 ± 2	49 ± 11	91 ± 17	48 ± 8
		333µg	14 ± 3	14 ± 3	71 ± 7	118 ± 1	43 ± 11
		1000µg	10 ± 2	27 ± 0	116 ± 5	146 ± 17	44 ± 4
		2500µg	11 ± 4	30 ± 3	180 ± 25	180 ± 7	51 ± 7
		5000µg	14 ± 2	41 ± 6	255 ± 8	186 ± 19	44 ± 11
	2 -AA	2.5µg	396 ± 25	143 ± 27	3757 ± 723	3936 ± 184
	2 -AA	10µg					328 ± 34

Key to positive controls:

NaN3: sodium azide

4 -NOPD: 4 -nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

2-AA: 2 -aminoanthracene

Applicant's summary and conclusion

Conclusions:

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 frameshifts in the genome of strains TA 1537 and TA 98 in the presence of metabolic activation (S9 mix).
Therefore, 3-Amino-4-methoxy-N-methylbenzamide is considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

The test item 3-Amino-4-methoxy-N-methylbenzamide was assessed for its potential to induce gene mutations according to the plate incorporation test usingSalmonellatyphimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and theEscherichia colistrain WP2 uvrA.

The assay was performed with and without rat liver microsomal 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

No precipitation of the test item occurred up to the highest investigated dose.

 

The plates incubated with the test item showed normal back­ground growth up to 5000 µg/plate with and without S9 mix in all strains used.

 

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.

Substantial increases in revertant colony numbers was observed following treatment with 3-Amino-4-methoxy-N-methylbenzamide in strains TA 1537 and TA 98 in the presence of metabolic activation (S9 mix). In strain 1537 the threshold of thrice the number of the corresponding solvent control was exceeded from 2500 to 5000 µg/plate. In strain TA 98 the threshold of twice the number of the corresponding solvent control was exceeded form 1000 to 5000 µg/plate.

A slight and dose dependent increase in the number of revertants was also detected in strain TA 100 with metabolic actvation (S9 mix) but the threshold of two was not reached.

 

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