Ethyl 4-[[(methylphenylamino)methylene]amino]benzoate

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:

From November 08, 2016 to December 28, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl 4-[[(methylphenylamino)methylene]amino]benzoate

EC Number:

260-976-0

EC Name:

Ethyl 4-[[(methylphenylamino)methylene]amino]benzoate

Cas Number:

57834-33-0

Molecular formula:

C17H18N2O2

IUPAC Name:

ethyl 4-[[(methylphenylamino)methylene]amino]benzoate

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Eversorb EP4
- Substance type: Liquid
- Composition of test material, percentage of components: 98.94%
- Lot/batch No.: 4023
- Storage condition of test material: Ambient

Method

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:

S9 Mix

Controls

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-Aminoanthracene

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Untreated negative controls validity:

valid

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:

not determined

Untreated negative controls validity:

valid

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:

not determined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 1. Characteristics of five Salmonella typhimurium strains

Test strain	Histidine requirement	uvrB mutation	rfa mutation	Ampicillin resistance
TA98	+	+	+	+
TA100	+	+	+	+
TA102	+	ϴ	+	+
TA1535	+	+	+	ϴ
TA1537	+	+	+	ϴ

+ means had the characteristic; ϴ means did not have the characteristic

Table 2. Toxicity of the test article of Salmonella typhimurium TA100

Group	Test article (mg/plate)	Reverse mutant colony number (CFU/plate)
With S9 Mix	Negative control group	236
	Positive control group^a	1532
	Test group
	5	264
	2.5	327
	1.25	283
	0.625	369
	0.3125	326
Without S9 Mix	Negative control group	182
	Positive control group	725
	Test group
	5	151
	2.5	185
	1.25	150
	0.625	154
	0.3125	164

^a Positive control group: With S9 Mix: 2-Aminoanthracene (4.0μg/plate).

Without S9 Mix: Sodium azide (5μg/plate).

 

Table 3. Reverse mutation test of Salmonella typhimurium TA98

Group	Test article (mg/plate)	Reverse mutant colony number (CFU/plate)			Average of colonies^a
		1	2	3
With S9 Mix	Negative control group	37	36	58	44 ± 12
	Positive control group^b	376	405	355	379 ± 25*
	Test group
	5	98	117	85	100 ± 16*
	2.5	71	87	81	80 ± 8
	1.25	59	42	37	46 ± 12
	0.625	44	44	72	53 ± 16
	0.3125	24	40	48	37 ± 12
Without S9 Mix	Negative control group	33	41	36	37 ± 4
	Positive control group	381	345	341	356 ± 22*
	Test group
	5	37	39	39	38 ± 1
	2.5	37	53	44	45 ± 8
	1.25	28	27	41	32 ± 8
	0.625	41	40	49	43 ± 5
	0.3125	30	48	36	38 ± 9

^a Average of colonies was shown as Mean ± S.D., the data were triplicate.             

^b Positive control group: With S9 Mix: Benzo [a] pyrene (4.0μg/plate).

Without S9 Mix: 4-Nitroquinoline-N-oxide (0.5μg/plate).

^*Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).

 

Table 4. Reverse mutation test of Salmonella typhimurium TA100

Group	Test article (mg/plate)	Reverse mutant colony number (CFU/plate)			Average of colonies^a
		1	2	3
With S9 Mix	Negative control group	216	221	243	277 ± 14
	Positive control group^b	1386	1535	1485	1469 ± 76*
	Test group
	5	232	322	363	306 ± 67
	2.5	249	253	249	250 ± 2
	1.25	214	198	269	227 ± 37
	0.625	240	271	259	257 ± 16
	0.3125	282	269	253	268 ± 15
Without S9 Mix	Negative control group	215	190	175	193 ± 20
	Positive control group	638	629	677	648 ± 26*
	Test group
	5	132	152	152	145 ± 12
	2.5	153	192	194	180 ± 23
	1.25	158	186	178	174 ± 14
	0.625	153	144	154	150 ± 6
	0.3125	166	250	248	221 ± 48

^a Average of colonies was shown as Mean ± S.D., the data were triplicate.

^b Positive control group: With S9 Mix: 2-Aminoanthracene (4.0μg/plate).

Without S9 Mix: Sodium azide (5μg/plate).

^*Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).

 

Table 5. Reverse mutation test of Salmonella typhimurium TA102

Group	Test article (mg/plate)	Reverse mutant colony number (CFU/plate)			Average of colonies^a
		1	2	3
With S9 Mix	Negative control group	482	416	464	454 ± 34
	Positive control group^b	945	968	984	966 ± 20*
	Test group
	5	437	446	472	452 ± 18
	2.5	470	426	453	450 ± 22
	1.25	512	489	478	493 ± 17
	0.625	498	473	515	495 ± 21
	0.3125	451	517	521	496 ± 39
Without S9 Mix	Negative control group	407	416	382	402 ± 18
	Positive control group	838	861	802	834 ± 30*
	Test group
	5	347	334	388	356 ± 28
	2.5	381	430	420	410 ± 26
	1.25	344	353	376	358 ± 17
	0.625	393	378	346	372 ± 24
	0.3125	443	391	371	402 ± 37

^a Average of colonies was shown as Mean ± S.D., the data were triplicate.

^b Positive control group: With S9 Mix: 2-Aminoanthracene (10μg/plate).

Without S9 Mix: Mitomycin C (0.5μg/plate).

^*Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).

 

Table 6. Reverse mutation test of Salmonella typhimurium TA1535

Group	Test article (mg/plate)	Reverse mutant colony number (CFU/plate)			Average of colonies^a
		1	2	3
With S9 Mix	Negative control group	23	33	24	27 ± 6
	Positive control group^b	363	348	325	345 ± 19*
	Test group
	5	32	25	45	34 ± 10
	2.5	15	23	20	19 ± 4
	1.25	12	18	16	15 ± 3
	0.625	20	17	15	17 ± 3
	0.3125	14	17	24	18 ± 5
Without S9 Mix	Negative control group	30	26	24	27 ± 3
	Positive control group	224	230	154	236 ± 16*
	Test group
	5	19	26	21	22 ± 4
	2.5	24	22	18	21 ± 3
	1.25	17	17	26	20 ± 5
	0.625	18	18	16	17 ± 1
	0.3125	17	28	35	27 ± 9

^a Average of colonies was shown as Mean ± S.D., the data were triplicate.

^b Positive control group: With S9 Mix: 2-Aminoanthracene (4.0μg/plate).

Without S9 Mix: Sodium azide (0.4μg/plate).

^*Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).

 

Table 7. Reverse mutation test of Salmonella typhimurium TA1537

Group	Test article (mg/plate)	Reverse mutant colony number (CFU/plate)			Average of colonies^a
		1	2	3
With S9 Mix	Negative control group	26	12	13	17 ± 8
	Positive control group^b	486	438	422	449 ± 33*
	Test group
	5	17	25	18	20 ± 4
	2.5	22	14	25	20 ± 6
	1.25	13	15	12	13 ± 2
	0.625	18	14	12	15 ± 3
	0.3125	11	12	13	12 ± 1
Without S9 Mix	Negative control group	17	15	19	17 ± 2
	Positive control group	1013	1036	874	974 ± 88*
	Test group
	5	6	8	6	7 ± 1
	2.5	5	11	7	8 ± 3
	1.25	8	2	6	5 ± 3
	0.625	2	5	7	5 ± 3
	0.3125	4	9	15	9 ± 6

^a Average of colonies was shown as Mean ± S.D., the data were triplicate.

^b Positive control group: With S9 Mix: 2-Aminoanthracene (4.0μg/plate).

Without S9 Mix: 9-Aminoacridine (50.0μg/plate).

^*Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).

Applicant's summary and conclusion

Conclusions:

According to OECD 471 test method, Eversorb EP4 was mutagenic in the reverse mutation analysis of Salmonella typhimurium.

Executive summary:

This test using the procedures outlined in the SuperLub Study Plan for M62-151100161001EN which is based on the SOP for the OECD 471 (SOPF-203) and OECD 471 (OECD, 1997). The results of this OECD 471 test for Eversorb EP4 show that test validity criteria was met.

Based on the preliminary assay results, 5mg/plate was set as the highest dose in this study. In the mutagenicity assay, five doses of Eversorb EP4 at 0.3125, 0.625, 1.25, 2.5 and 5mg/plate, concurrent negative and strain-specific positive controls were tested in tester strains TA98, TA100, TA102, TA1535 and TA1537 in triplicate with or without S9 Mix activation. At the highest dose with S9 Mix in tester strains of Salmonella typhimurium TA98 of test groups, the results showed that the number of revertants increase twice more than negative controls group. Based on the data obtained from this study, it was concluded that under the test condition, Eversorb EP4 was mutagenic in the reverse mutation analysis of Salmonella typhimurium.