3-[ethyl[4-[(4-nitrophenyl)azo]phenyl]amino]propiononitrile

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:

30 March 2017 to 13 April 2017

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:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: 'Standards for Mutagenicity Tests using Microorganisms' (Notification No. 77, September 1, 1988 & partial revision: Notification No. 67, June 2, 1997, Ministry of Labour, Japan and Notification No. 120, December 25, 2000, Ministry of Labour, Japan)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: "JAPAN: "Guidelines for Toxicity Testing of New Chemical Substances"

Version / remarks:

(Notification No. 7 of 0331 Pharmaceutical and Food safety Bureau, MInistry of Health, Labour and Welfare, No. 5 of the Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry, and No. 110331009 of Environmental Policy Bureau, Ministry of the Environment, Japan, 31 Macrh 2011)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-[ethyl[4-[(4-nitrophenyl)azo]phenyl]amino]propiononitrile

EC Number:

250-654-8

EC Name:

3-[ethyl[4-[(4-nitrophenyl)azo]phenyl]amino]propiononitrile

Cas Number:

31482-56-1

Molecular formula:

C17H17N5O2

IUPAC Name:

3-[ethyl[4-[(4-nitrophenyl)azo]phenyl]amino]propiononitrile

Test material form:

solid: particulate/powder

Method

Target gene:

Salmonella typhimurium
TA1537 (frame shift mutations)
TA98 (frame shift mutations)
TA1535 (base-pair mutations)
TA100 (base-pair mutations)

Escherichia coli
WP2 uvrA (base-pair substitution)

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:

S9 mix

Test concentrations with justification for top dose:

Preliminary test - 1.2, 4.9, 20, 78, 313, 1250 and 5000 µg/plate.

In the preliminary test, the growth inhibition by the test material was not observed in any strains either with or without metabolic activation. An increase in the number of revertant colonies more than twice that of the negative control was observed in S. typhimurium TA strains without metabolic activation and all strains with metabolic activation. Test material precipitate was observed at 5000 µg/plate both with and without metabolic activation.

5000 µg/plate was selected as the highest dose in the main tests for S. typhimurium TA 1535 without metabolic activation and E. coli WP2 uvraA both with and without metabolic activation. This dose was diluted four times (using a common ratio of 2) to provide a total of five doses. 5000 µg/plate was selected as the highest dose for S. typhimurium TA100 and TA98 without metabolic activation and diluted 8 times (using a common ratio of 2) to provide a total of 9 doses. 5000 µg/plate was also selected for S. typhimurium TA1537 without metabolic activation and S. typhimurium TA100, TA1535 and TA98 with metabolic activation and diluted 10 times (using a common ration of 2) to provide a total of 11 doses.

Doses tested are detailed in the results section.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: N,N-dimethylformamide (DMF)
- Justification for choice of solvent/vehicle: the test material could be suspended in DMF at 100 mg/mL and dissolved at 50 mg/mL.

Controls

Untreated negative controls:

yes

Remarks:

DMF

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

furylfuramide

other: 2-aminoanthracene and 2-methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine·2HCl

Details on test system and experimental conditions:

TEST PROCEDURES
For the tests without metabolic activation, 0.5 mL of 0.1 M Na-phosphate buffer (pH 7.4) and 0.1 mL of each fresh bacterial culture were added to each tube containing 0.05 mL of the test solution or the negative control solution. For the tests with metabolic activation, 0.5 mL of the S9 mix was added to each tube instead of the buffer. The mixture was pre-incubated in a water bath at 37°C for 20 minutes while shaking horizontally and then 2 mL of top agar was added to the mixture and the contents of each tube were poured over the surface of the minimal glucose agar plate. The same test with 0.1 mL of the positive control solution was also performed.

One minimal glucose agar plate was used for each dose level in the preliminary test and three minimal glucose agar plates were used for each dose level in the two main tests which were performed at the same doses.

For the sterility test, 0.05 mL of the test solution of the maximum concentration and 0.5 mL of the S9 mix were put into each tube, 2 mL of top agar was then added to the tube and the contents of each tube were poured over the surface of the minimal glucose agar plates.

As top agar, the 0.5 mM biotin / 0.5 mM L-histidine solution and the 0.05 mM L-tryptophan solution were added to the soft agar solution (0.6% agar and 0.5% NaCl) by volume of 1/10 for the S. typhimurium TA strains and the E. coli strain, respectively.

All plates were incubated at 37°C for 48 hours and the number of revertant colonies was counted. Afterwards, growth inhibition of the test strains was checked using a stereoscopic microscope.

COUNTING PROCEDURES
The number of revertant colonies was counted visually due to the precipitate of the test material on the plates. The revertant colonies of the positive controls were counted with a colony counter.

Evaluation criteria:

In the main tests, if the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates (based on twice as many as the negative control), and dose-response and reproducibility were also observed, the test material was to be judged positive. The results at each concentration were demonstrated with the mean and standard deviation.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the main tests, a dose-dependent increase in the number of revertant colonies, more than twice that of the negative control, and reproducibility were observed in S. typhimurium TA100, TA98 and TA1537 without metabolic activation and all strains with metabolic activation. An increase in the number of revertant colonies was observed in S. typhimurium TA5135 without metabolic activation and was more than twice that of the negative control in the first main test.

The revertant colonies of the positive controls showed an increase of more than twice that of the negative controls and they were within the appropriate limits of the historical control data indicating that the study was valid.

The test material was considered to be positive for mutagenicity.

The growth inhibition of the test strains by the test material was not observed. Test material precipitate on plates was observed at doses of 2500 µg/plate and higher both with and without metabolic activation.

No growth of the bacteria was observed in the sterility test.

Any other information on results incl. tables

Preliminary test results

		Number of revertant colonies/plate
With or without S9 mix	Dose (µg/plate)	TA100	TA1535	WP2 uvr A	TA98	TA1537
-S9 mix	Solvent control	107	8	22	30	8
	1.2	109	6	23	24	6
	4.9	118	8	29	38	13
	20	137	9	26	28	23
	78	190	11	29	63	39
	313	245	13	20	139	90
	1250	315	13	21	313	156
	5000*	444	20	18	715	247
+S9 mix	Solvent control	135	16	34	35	19
	1.2	115	16	29	43	15
	4.9	155	13	28	53	14
	20	258	44	21	278	23
	78	295	45	31	371	66
	313	386	43	49	631	210
	1250	856	38	108	1321	1000
	5000*	1405	64	172	1913	1127
Positive control without S9 mix	Name   (Dose, µg/plate)	AF-2 (0.01)	NaN3  (0.5)	AF-2 (0.01)	AF-2 (0.1)	ICR-191 (1.0)
	Number of colonies/plate	526	464	171	509	1176
	Name (Dose, µg/plate)	B[α]P (5.0)	2AA (2.0)	2AA (10.0)	B[α]P (5.0)	B[α]P (5.0)
	Number of colonies/plate	779	340	409	226	57

AF-2: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide

NaN_3: sodium azide

ICR-191: 2-methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylaminoacridine · 2HCl

B[α]P: Benzo [α] pyrene

2AA: 2-aminoanthracene

* the precipitate of the test material on the plates was observed

NT = not tested

First main test results

		Number of revertant colonies/plate
With or without S9 mix	Dose (µg/plate)	TA100	TA1535	WP2 uvr A	TA98	TA1537
-S9 mix	Solvent control	111 ± 5.0	13 ± 2.0	29 ± 2.5	34 ± 2.3	8 ± 2.0
	4.9	NT	NT	NT	NT	11 ± 1.2
	10	NT	NT	NT	NT	16 ± 3.5
	20	124 ± 17.8	NT	NT	50 ± 9.0	20 ± 3.0
	39	142 ± 8.1	NT	NT	82 ± 9.3	36 ± 6.9
	78	162 ± 5.1	NT	NT	113 ± 2.9	49 ± 5.1
	156	199 ± 21.3	NT	NT	172 ± 21.7	76 ± 6.1
	313	237 ± 25.4	17 ± 0.6	23 ± 4.6	249 ± 18.0	96 ± 11.0
	625	297 ± 10.2	20 ± 2.0	26 ± 1.5	299 ± 12.1	138 ± 8.7
	1250	321 ± 3.5	24 ± 2.5	24 ± 1.5	442 ± 22.5	172 ± 8.9
	2500*	394 ± 26.4	20 ± 1.7	27 ± 3.5	699 ± 25.8	260 ± 28.6
	5000*	523 ± 18.8	27 ± 1.0	25 ± 2.3	1008 ± 28.8	318 ± 16.8
Positive control without S9 mix	Name	AF-2	NaN3	AF-2	AF-2	ICR-191
	Dose (µg/plate)	0.01	0.5	0.01	0.1	1.0
	Number of colonies/plate	537 ± 12.9	426 ± 17.0	136 ± 10.4	536 ± 16.6	1295 ± 73.5
+S9 mix	Solvent control	124 ± 10.2	14 ± 2.9	34 ± 4.0	40 ± 3.1	17 ± 2.1
	4.9	142 ± 9.0	14 ± 2.5	NT	58 ± 1.7	NT
	10	211 ± 6.2	20 ± 3.0	NT	178 ± 24.3	NT
	20	287 ± 14.4	38 ± 4.6	NT	313 ± 23.1	26 ± 2.1
	39	331 ± 29.3	48 ± 4.9	NT	399 ± 8.7	35 ± 3.6
	78	315 ± 18.9	50 ± 3.5	NT	421 ± 21.7	64 ± 3.2
	156	388 ± 13.0	46 ± 5.7	NT	578 ± 41.9	118 ± 10.0
	313	452 ± 8.2	47 ± 4.6	46 ± 5.6	699 ± 19.1	229 ± 10.1
	625	656 ± 41.1	37 ± 3.6	79 ± 5.0	1191 ± 120.4	465 ± 34.2
	1250	983 ± 22.9	47 ± 10.4	130 ± 12.2	1521 ± 74.6	847 ± 72.1
	2500*	1465 ± 53.9	53 ± 6.0	167 ± 4.0	1861 ± 28.0	1217 ± 65.7
	5000*	1591 ± 61.3	55 ± 2.6	192 ± 4.2	2149 ± 134.0	1221 ± 72.2
Positive control with S9 mix	Name	B[α]P	2AA	2AA	B[α]P	B[α]P
	Dose (µg/plate)	5.0	2.0	10.0	5.0	5.0
	Number of colonies/plate	878 ± 64.1	318 ± 18.1	416 ± 44.1	237 ± 7.8	72 ± 8.7

AF-2: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide

NaN_3: sodium azide

ICR-191: 2-methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylaminoacridine·2HCl

B[α]P: Benzo [α] pyrene

2AA: 2-aminoanthracene

* the precipitate of the test material on the plates was observed

NT = not tested

Second main test results

		Number of revertant colonies/plate
With or without S9 mix	Dose (µg/plate)	TA100	TA5135	WP2 uvr A	TA98	TA1537
-S9 mix	Solvent control	96 ± 13.5	14 ± 0.0	22 ± 2.3	24 ± 3.2	8 ± 1.5
	4.9	NT	NT	NT	NT	13 ± 3.2
	10	NT	NT	NT	NT	18 ± 2.1
	20	115 ± 5.0	NT	NT	43 ± 3.1	22 ± 2.0
	39	125 ± 7.2	NT	NT	60 ± 2.1	28 ± 2.6
	78	145 ± 8.4	NT	NT	83 ± 11.1	41 ± 3.1
	156	176 ± 15.4	NT	NT	114 ± 9.8	83 ± 7.4
	313	230 ± 14.8	13 ± 2.1	22 ± 2.1	167 ± 28.6	93 ± 14.7
	625	252 ± 17.1	13 ± 2.3	20 ± 0.6	246 ± 22.2	123 ± 16.0
	1250	268 ± 24.9	18 ± 3.2	21 ± 1.7	331 ± 5.5	181 ± 23.6
	2500*	340 ± 22.1	20 ± 4.5	26 ± 2.6	468 ± 10.7	216 ± 15.3
	5000*	438 ± 22.0	22 ± 2.1	25 ± 1.2	731 ± 26.5	307 ± 23.2
Positive control without S9 mix	Name	AF-2	NaN3	AF-2	AF-2	ICR-191
	Dose (µg/plate)	0.01	0.5	0.01	0.1	1.0
	Number of colonies/plate	531 ± 17.1	425 ± 9.6	128 ± 19.8	516 ± 4.6	1408 ± 241.3
+S9 mix	Solvent control	118 ± 11.0	13 ± 1.0	3.7 ± 3.1	39 ± 2.3	14 ± 0.6
	4.9	123 ± 15.3	17 ± 1.2	NT	67 ± 9.5	NT
	10	185 ± 1.2	23 ± 4.9	NT	191 ± 7.8	NT
	20	253 ± 22.2	32 ± 3.0	NT	263 ± 25.0	25 ± 3.6
	39	248 ± 9.2	46 ± 4.2	NT	301 ± 18.3	38 ± 5.7
	78	274 ± 17.4	41 ± 5.1	NT	357 ± 48.7	62 ± 8.0
	156	294 ± 9.0	40 ± 5.5	NT	502 ± 34.6	116 ± 3.2
	313	391 ± 13.5	44 ± 6.6	51 ± 4.2	678 ± 34.0	236 ± 14.2
	625	501 ± 3.6	39 ± 2.3	76 ± 2.3	905 ± 16.9	517 ± 28.1
	1250	849 ± 37.3	51 ± 4.0	110 ± 0.6	1360 ± 175	886 ± 54.3
	2500*	1211 ± 45.4	54 ± 3.1	185 ± 9.7	1587 ± 47.9	1077 ± 27.1
	5000*	1420 ± 2.0	61 ± 9.0	212 ± 21.5	2160 ± 160.5	1101 ± 26.4
Positive control with S9 mix	Name	B[α]P	2AA	2AA	B[α]P	B[α]P
	Dose (µg/plate)	5.0	2.0	10.0	5.0	5.0
	Number of colonies/plate	787 ± 51.4	288 ± 24.9	338 ± 41.6	189 ± 13.0	60 ± 6.7

AF-2: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide

NaN_3: sodium azide

ICR-191: 2-methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylaminoacridine·2HCl

B[α]P: Benzo [α] pyrene

2AA: 2-aminoanthracene

* the precipitate of the test material on the plates was observed

NT = not tested

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the test material was determined to be mutagenic in the bacterial reverse mutation assay.

Executive summary:

The mutagenicity potential of the test material was assessed with Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WPA uvrA following the standardised guidelines OECD 471, the Japanese Standards for Mutagenicity Tests using Microorganisms, and the Japanese Guidelines for Toxicity Testing of New Chemical Substances. The study was conducted under GLP conditions.

In the two main tests, a dose-dependent increase in the number of revertant colonies, more than twice that of the negative control, and reproducibility was observed in S. typhimurium TA100, TA98 and TA5137 without metabolic activation and in all strains with metabolic activation. It is concluded that the test material is mutagenic to bacteria under the study conditions.