N-(2-(4-amino-N-ethyl-m-toluidino)ethyl)methanesulphonamide sesquisulphate

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:

18 Jan 2018 - 07 Feb 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-(2-(4-amino-N-ethyl-m-toluidino)ethyl)methanesulphonamide sesquisulphate

EC Number:

247-161-5

EC Name:

N-(2-(4-amino-N-ethyl-m-toluidino)ethyl)methanesulphonamide sesquisulphate

Cas Number:

25646-71-3

Molecular formula:

C12H21N3O2S.3/2H2O4S

IUPAC Name:

bis(N-{2-[(4-amino-3-methylphenyl)(ethyl)amino]ethyl}methanesulfonamide); tris(sulfuric acid)

Method

Target gene:

his operon for S. typhimurium strains
trp operon for 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 Arocolor 1254.

Test concentrations with justification for top dose:

Pre-experiment (dose-range finding test, Direct Plate Assay): 1.7, 5.4, 17, 52, 164, 512, 1600, 5000 µg/plate
Main-experiment (Pre-incubation Assay): 17, 52, 164, 512, 1600, 5000 µg/plate (Based on the results of the dose-range finding test)

Vehicle / solvent:

- Vehicle/solvent used: water (Milli-Q-water, Millipore Corp., Bedford, MA., USA).
- Justification for choice of solvent/vehicle: The test item formed a clear colorless solution in Milli-Q water.

- Vehicle/solvent used for positive control items: Saline = physiological saline (Eurovet Animal Health, Bladel, The Netherlands)
DMSO = dimethyl sulfoxide (Merck, Darmstadt, Germany)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191, 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar, Direct Plate Assay, Pre-incubation assay

DURATION
- Preincubation period: 30 ± 2 min
- Exposure duration: at least 48 ± 4 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.

Evaluation criteria:

ACCEPTABILITY CRITERIA
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) 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.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

EVALUATION CRITERIA
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment. A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

Mean values and standard deviations were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed at 1600 μg/plate onwards without metabolic activation and at 5000 μg/plate with metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

2.3-fold dose-related increase in the number of revertant colonies in the absence of S9-mix.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed at 1600 μg/plate onwards without metabolic activation and at 5000 μg/plate with metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

1.9-fold dose-related increase in the number of revertant colonies in the presence of S9-mix. The increase observed was within the laboratory historical control data ranges and less than two-fold the concurrent solvent control

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed at 1600 μg/plate onwards without metabolic activation and at 5000 μg/plate with metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed at 1600 μg/plate onwards without metabolic activation and at 5000 μg/plate with metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed at 1600 μg/plate onwards without metabolic activation and at 5000 μg/plate with metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Direct Plate Assay
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance on the plates was not observed at the start or at the end of the incubation period in any tester strain.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Cytotoxicity, as evidenced by a decrease in the number of revertants and/or reduction of the bacterial background lawn, was observed in all tester strains. Except for tester strain WP2uvrA, where no toxicity was observed in the absence or presence of S9-mix.

Pre-Incubation Assay
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance on the plates was not observed at the start or at the end of the incubation period in any tester strain.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Cytotoxicity, as evidenced by a decrease in the number of revertants and/or reduction of the bacterial background lawn, was observed in all tester strains from 1600 μg/plate onwards without metabolic activation and at 5000 μg/plate with metabolic activation. Except for tester strain WP2uvrA in the presence of S9-mix, where no toxicity was observed

Any other information on results incl. tables

  Table 3: Dose-Range Finding Test (Direct-Plate assay)

	EXPERIMENT 1 (Revertant colonies per plate ± SD)
	S9-Mix	Without
	Test item (µg/plate)	TA 100	WP2uvrA
Vehicle Control		113 ± 11	33 ± 11
Test Substance	1.7	121 ± 15	25 ± 5
	5.4	123 ± 19	37 ± 2
	17	115 ± 12	21 ± 1
	52	134 ± 12	33 ± 6
	164	141 ± 13	37 ± 6
	512	166 ± 16	35 ± 0
	1600	292 ± 22^n	41 ± 9
	5000	0 ± 0^aNP	51 ± 4^nNP
Positive Control		744 ± 65	1602 ± 77
	S9-Mix	With
	Test item (µg/plate)	TA 100	WP2uvrA
Vehicle Control	-	118 ± 6	43 ± 7
Test Substance	1.7	100 ± 8	50 ± 6
	5.4	106 ± 13	40 ± 3
	17	116 ± 13	35 ± 9
	52	122 ± 6	38 ± 8
	164	135 ± 18	43 ± 4
	512	169 ± 21	58 ± 5
	1600	310 ± 19	75 ± 4
	5000	106 ± 94^1NP	52 ± 5^nNP
Positive Control		2156 ± 227	558 ± 54
	1=Revertants were only observed on a small part of the plate with normal background, no bacterial growth or background was observed on a large part of the plate NP= No precipitate a = Bacterial background lawn absent n = Normal bacterial background lawn s = Bacterial background lawn slightly reduced

 

Table 4: Direct-Plate assay

	EXPERIMENT 1 (Revertant colonies per plate ± SD)
	S9-Mix	Without
	Test item (µg/plate)	TA 1535	TA 1537	TA 98
Vehicle Control		12 ± 2	3 ± 2	11 ± 1
Test Substance	17	12 ± 2	3 ± 0	17 ± 5
	52	13 ± 5	4 ± 3	14 ± 3
	164	14 ± 4	2 ± 1	11 ± 3
	512	13 ± 2	2 ± 1	10 ± 6
	1600	11 ± 3^n	2 ± 1^n	12 ± 6^n
	5000	0 ± 0^aNP	2 ± 2^eNP	0 ± 0^aNP
Positive Control		1091 ± 65	834 ± 49	1141± 30
	S9-Mix	With
	Test item (µg/plate)	TA 1535	TA 1537	TA 98
Vehicle Control	-	11 ± 6	3 ± 4	16 ± 2
Test Substance	17	12 ± 5	4 ± 3	16 ± 3
	52	10 ± 2	7 ± 4	22 ± 6
	164	13 ± 5	4 ± 1	16 ± 4
	512	17 ± 8	3 ± 2	17 ± 4
	1600	18 ± 3	3 ± 2	19 ± 7
	5000	5 ± 8^1NP	1 ± 2^1NP	3 ± 2^1NP
Positive Control		408 ± 32	374± 108	1298± 195
	1=Revertants were only observed on a small part of the plate with normal background, no bacterial growth or background was observed on a large part of the plate NP= No precipitate a = Bacterial background lawn absent n = Normal bacterial background lawn s = Bacterial background lawn slightly reduced

 

Table 5: Plate-Incubation Test

	EXPERIMENT 2 (Revertant colonies per plate ± SD)
	S9-Mix	Without
	Test item (µg/plate)	TA 1535	TA 1537	TA 98	TA 100 (fold)	WP2uvrA (fold)
Vehicle Control		10 ± 2	3 ± 4	18 ± 3	106 ± 14	19 ± 5
Test Substance	17	13 ± 5	3 ± 1	17 ± 6	122 ± 15	28 ± 2
	52	12 ± 2	3 ± 2	13 ± 5	130 ± 5	29 ± 15 (1.5)
	164	19 ± 1	3 ± 2	14 ± 3	134 ± 16 (1.3)	35 ± 7 (1.8)
	512	13± 3^n	8 ± 2^n	17 ± 3^n	195 ± 5^n(1.8)	44 ± 8^n(2.3)
	1600	0 ± 0^a	0± 0^a	0± 0^a	0± 0^a	1± 0^1n
	5000	0 ± 0^aNP	0 ± 0^aNP	0 ± 0^aNP	0 ± 0^aNP	0 ± 0^aNP
Positive Control		1036 ± 33	138 ± 14	1146 ± 68	781 ± 11	152 ± 85
	S9-Mix	With
	Test item (µg/plate)	TA 1535	TA 1537	TA 98	TA 100	WP2uvrA
Vehicle Control	-	11 ± 2	5 ± 3	19 ± 6	102 ± 2	30 ± 7
Test Substance	17	13 ± 2	4 ± 2	15 ± 6	166 ± 18	36 ± 6
	52	10 ± 5	7 ± 2	13 ± 2	180 ± 20	42 ± 6
	164	14 ± 3	7 ± 2	19 ± 4	139 ± 7	39 ± 8
	512	10 ± 0	5 ± 3^n	13 ± 2	190 ± 26 (1.9)	42 ± 4 (1.4)
	1600	14 ± 6	2 ± 1^s	15 ± 3^n	304 ± 22^n(3.0)	56 ± 13 (1.9)
	5000	0 ± 1^1nNP	0 ± 0^aNP	0 ± 0^aNP	0 ± 0^aNP	57 ± 11^nNP(1.9)
Positive Control		247 ± 22	230 ± 32	762 ± 14	1586 ± 89	587 ± 30
	1=Revertants were only observed on a small part of the plate with normal background, no bacterial growth or background was observed on a large part of the plate NP= No precipitate a = Bacterial background lawn absent n = Normal bacterial background lawn s = Bacterial background lawn slightly reduced

 

Applicant's summary and conclusion

Conclusions:

Under the conditions of the conducted test the substance induced significant dose-related increases in the number of revertant colonies in the tester strain TA100 and WP2uvrA both in the absence and presence of S9-mix. In the latter, only in the absence of S-9 mix in experiment 2, these increases exceeded the two-fold threshold (2.3-fold). Based on these results, the test substance is regarded to be mutagenic in bacteria.