N-[2-[(2,6-dicyano-4-nitrophenyl)azo]-5-(diethylamino)phenyl]acetamide

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:

October 28th 1998 to October 30th 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guideline For Testing of Chemicals, 471 Bacterial Reverse Mutation Test Adopted: July 21st, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

U.S. EPA: OPPTS 870.5100 Health Effects Test Guidelines Bacterial Reverse Mutation Test, August 1998

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

EEC Directive 92169, L 383 A, Annex B 14
EEC Directive 92169, L 383 A, Annex B 13

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-[2-[(2,6-dicyano-4-nitrophenyl)azo]-5-(diethylamino)phenyl]acetamide

EC Number:

255-473-8

EC Name:

N-[2-[(2,6-dicyano-4-nitrophenyl)azo]-5-(diethylamino)phenyl]acetamide

Cas Number:

41642-51-7

Molecular formula:

C20H19N7O3

IUPAC Name:

N-[2-[(2,6-dicyano-4-nitrophenyl)azo]-5-(diethylamino)phenyl]acetamide

Test material form:

solid: particulate/powder

Details on test material:

Disperse Blue 165

Specific details on test material used for the study:

No further details specified in the study report.

Method

Target gene:

Histidine and tryptophan

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

plate Incorporation test:
a: without metabolic activation:
50, 160, 500, 1600 and 5000 μg/plate
b: with metabolic activation:
50, 160, 500, 1600 and 5000 μg/plate

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 1-methyl-3-nitro-1-nitrosoguanidine (MNNG); 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

Experimental conditions in vitro: approx. 37 °C in an incubator

Bacteria
The strains of Salmonella typhimurium were obtained from Professor B.N. Ames, University of California, U.S.A. The strain of E. coli was obtained from the National Collection of Industrial Bacteria, Aberdeen, Scotland.
Bacteria were grown overnight in nutrient broth (25 g Oxoid Nutrient Broth No. 2 /liter) at approx. 37 °C. The amount of bacteria in the cell suspension was checked by nephelometry. Inoculation was performed with stock cultures which had been stored at approx. -80 °C. The different bacterial strains are checked half-yearly with regard to their respective biotin, histidine and/or tryptophan requirements, membrane permeability, ampicillin resistance, crystal violet sensitivity, UV resistance and response to diagnostic mutagens. All criteria for a valid assay were fulfilled as described.

Assay procedure
The mutation test (plate incorporation test) was performed in both the presence and absence of S9-mix using all bacterial tester strains and a range of concentrations of the test substance. Positive and negative controls as well as solvent controls were included in each test. Triplicate plates were used.
The mutation experiment also assessed the toxicity of the test substance by evaluation of the bacterial lawn in order to select a suitable range of dose levels for a second mutation test. The highest concentration was usually 50 mg/ml of the test substance in the chosen solvent, which provided a final concentration of 5000 μg/plate. Further dilutions of 1600, 500, 160 and 50 μg/plate were used.
A reduced rate of spontaneously occurring colonies and visible thinning of the bacterial lawn were used as toxicity indicators. Thinning of the bacterial lawn was evaluated microscopically.

If the total number of concentrations selected for evaluation in the plate incorporation test does not allow for a statement of genotoxicity of the test substance to be made, an additional plate incorporation test, based on the toxicity results of the first test, has to be performed. If negative or equivocal results obtained, a second mutation experiment was performed on the basis of toxicity results in the plate incorporation test as a pre-incubation test.

For mutagenicity testing top agar was prepared for the Salmonella strains by mixing 100 ml agar (0.6% (w/v) agar, 0.5% (w/v) NaCI) with 10 ml of a 0.5 mM histidine-biotin solution. With E. coli histidine was replaced by tryptophan (2.5 ml, 0.5 mM). The following ingredients were added (in the following order) to 2 ml of molten top agar at approx. 48 °C:
0.5 ml S9-mix (if required) or buffer
0.1 ml of an overnight nutrient broth culture of the bacterial tester strain
0.1 ml test compound suspension (suspended in DMSO)

After mixing, the liquid was poured into a petri dish containing a 25 ml layer of minimal agar (1 .5% (w/v) agar, Vogel-Bonner E medium with 2% (w/v) glucose). After incubation for approximately 48 hours at approx. 37 °C in the dark, colonies (his+ and trp+ revertants) were counted with an automatic colony counter (Artec counter Model 880). The counter was calibrated for each test by comparison of manual count data of three control plates with automatic data of the colony counter.
A correction factor was determined to compensate for differences between manual and automatic count. This correction factor was used to automatically adjust the observed number of colonies on each plate to more accurately reflect the actual number of colonies present.

Rationale for test conditions:

In accordance with the test guidelines.

Evaluation criteria:

Criteria for a valid assay
The assay is considered valid if the following criteria are met:
- the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
- the positive controls induced increases in the mutation frequency which were both statistically significant and within the laboratory's normal range

Criteria for a positive response
A test compound is classified as mutagenic if it has either of the following effects:
a) it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn.

If the test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system.

Statistics:

Not specified.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test compound proved to be toxic at the dose of 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test compound proved to be toxic at the dose of 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test compound proved to be toxic at the dose of 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test compound proved to be toxic at the dose of 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 8

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 9

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

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:

Sterility checks and control plates
Sterility of S9-mix and the test compound were indicated by the absence of contamination on the test material and S9-mix sterility check plates. Control plates {background control and positive controls) gave the expected number of colonies, i.e. values were within the laboratory's historical control range.

Solubility and toxicity
The test compound was suspended in DMSO and a stock solution of 50 mg/ml was prepared for the highest concentration, which provided a final concentration of 5000 μg/plate. Further dilutions of 1600, 500, 160 and 50 μg/plate were used in the plate incorporation test.
Visible precipitation of the test compound on the plates was observed at 500 μg/plate and above.
The test compound proved to be not toxic to the bacterial strains in the mutagenicity experiment in the presence of metabolic activation. No toxicity was observed at the strain Escherichia coli WP 2uvrA with and without S9-mix.
The test compound proved to be toxic to the Salmonella strains at the dose of 5000 μg/plate in this plate incorporation test in the absence of metabolic activation.

Mutagenicity
The number of colonies per plate with each strain as well as mean values of 3 plates are given.
In the absence and presence of the metabolic activation system the test compound induced a significant and dose-dependent increase in the number of revertant colonies with the Salmonella strains. The Eschericia coli strain WP 2uvrA showed no significant increases in the number of revertant colonies.
All positive controls produced significant increases in the number of revertant colonies.
Thus, the sensitivity of the assay and the efficacy of the exogenous metabolic activation system were demonstrated.

Any other information on results incl. tables

***CONTROLS***

SOLVENT CONTROL COMMENTS: NONE

NEGATIVE CONTROL COMMENTS: NONE

POSITIVE CONTROL COMMENTS: NONE

 

STRAIN		DOSE LEVELS (μg/plate)	MEAN	STANDARD DEVIATION	RATIO: TEST/ CONTROL	BACTERIAL LAWN	NO REVERTANTS/PLATE
							PLATE 1	PLATE 2	PLATE 3
TA 100	+S9	SOLVENT CONTROLS
			107.7	5.0			103	107	113
		NEGATIVE CONTROLS
			135.0	3.0	1.3		132	135	138
		POSITIVE CONTROLS				2-AMINOANTHRACENE
		0.5	669.0	25.5	6.2		694	643	670
TA 100	-S9	SOLVENT CONTROLS
			140.0	4.4			135	142	143
		NEGATIVE CONTROLS
			115.7	20.4	0.8		139	101	107
		POSITIVE CONTROLS				SODIUM-AZIDE
		1.0	540.0	49.5	3.9		597	515	508
TA 1535	+S9	SOLVENT CONTROLS
			5.7	2.1			5	4	8
		NEGATIVE CONTROLS
			4.7	2.3	0.8		2	6	6
		POSITIVE CONTROLS				2-AMINOANTHRACENE
		1.0	164.0	3.5	28.8		160	166	166
TA 1535	-S9	SOLVENT CONTROLS
			5.7	1.5			4	7	6
		NEGATIVE CONTROLS
			7.0	2.6	1.2		10	5	6
		POSITIVE CONTROLS				SODIUM-AZIDE
		1.0	308.7	37.6	54.2		305	273	348
TA 1537	+S9	SOLVENT CONTROLS
			3.7	2.1			3	6	2
		NEGATIVE CONTROLS
			4.3	1.2	1.2		5	3	5
		POSITIVE CONTROLS				2-AMINOANTHRACENE
		1.0	136.3	13.1	36.8		124	135	150
TA 1537	-S9	SOLVENT CONTROLS
			5.3	1.5			7	4	5
		NEGATIVE CONTROLS
			4.0	1.0	0.8		5	3	4
		POSITIVE CONTROLS				9-AMINOACRIDINE
		50.0	96.3	9.0	18.2		101	102	86
TA 98	+S9	SOLVENT CONTROLS
			15.3	5.1			14	11	21
		NEGATIVE CONTROLS
			19.3	4.5	1.3		15	24	19
		POSITIVE CONTROLS				2-AMINOANTHRACENE
		0.5	548.3	98.5	35.8		532	459	654
TA 98	-S9	SOLVENT CONTROLS
			15.3	0.6			15	16	15
		NEGATIVE CONTROLS
			13.7	3.1	0.9		17	11	13
		POSITIVE CONTROLS				2-NITROFLUORENE
		2.5	459.7	28.1	30.0		489	457	433
WP2uvrA	+S9	SOLVENT CONTROLS
			21.7	2.3			19	23	23
		NEGATIVE CONTROLS
			18.0	6.1	0.8		14	15	25
		POSITIVE CONTROLS				2-AMINOANTHRACENE
		30.0	157.3	20.3	7.2		151	141	180
WP2uvrA	-S9	SOLVENT CONTROLS
			17.0	2.0			19	17	15
		NEGATIVE CONTROLS
			24.0	1.0	1.4		25	23	24
		POSITIVE CONTROLS				MNNG
		4.0	115.3	14.5	6.8		106	108	132

 

***TEST***

COMMENTS:   ALL STERILITY CONTROL PLATES WERE STERILE.

                       VISIBLE PRECIPITATION OF THE TEST COMPOUND ON THE PLATES WAS OBSERVED AT 500 MICROGRAM/PLATE AND ABOVE.

STRAIN		DOSE LEVEL (μg/plate)	MEAN	STANDARD DEVIATION	RATIO: TEST/ CONTROL	BACTERIAL LAWN	NO REVERTANTS/PLATE
							PLATE 1	PLATE 2	PLATE 3
TA 100	+S9	0. 50. 160. 500. 1600. 5000.	107.7 813.7 854.3 838.7 795.3 873.7	5.0 59.7 32.3 67.5 51.1 37.2	7.6 7.9 7.8 7.4 8.1		103 762 880 912 832 831	107 879 865 779 737 891	113 800 818 825 817 899
TA 100	-S9	0. 50. 160. 500. 1600. 5000.	140.0 548.7 546.0 583.3 620.7 1034.3	4.4 112.2 84.1 54.5 290.6 48.5	3.9 3.9 4.2 4.4 7.4	INCOMPLETE	135 447 511 633 442 1012	142 530 485 592 464 1090	143 669 642 525 956 1001
TA 1535	+S9	0. 50. 160. 500. 1600. 5000.	5.7 60.0 85.7 113.3 78.3 110.0	2.1 6.1 33.8 45.5 0.6 14.9	10.5 15.0 19.9 13.7 19.3		5 56 73 115 78 116	4 57 125 158 79 121	8 67 60 67 78 93
TA 1535	-S9	0. 50. 160. 500. 1600. 5000.	5.7 34.0 16.7 19.3 35.7 58.7	1.5 3.0 2.3 2.3 12.2 4.0	6.0 2.9 3.4 6.3 10.3	INCOMPLETE	4 37 18 18 25 58	7 34 18 18 33 63	6 31 14 22 49 55
TA 1537	+S9	0. 50. 160. 500. 1600. 5000.	3.7 246.7 221.7 183.7 318.3 394.0	2.1 55.9 45.2 13.3 64.0 6.9	66.7 59.9 49.6 86.0 106.5		3 210 273 176 287 386	6 219 204 199 392 398	2 311 188 176 276 398
TA 1537	-S9	0. 50. 160. 500. 1600. 5000.	5.3 44.0 33.3 42.7 78.0 194.0	1.5 15.7 7.2 8.0 10.5 25.2	8.3 6.3 8.1 14.7 36.6	INCOMPLETE	7 61 38 35 77 207	4 41 37 51 68 165	5 30 25 42 89 210
TA 98	+S9	0. 50. 160. 500. 1600. 5000.	15.3 835.3 809.7 1030.7 1501.0 1661.0	5.1 158.8 138.5 73.4 367.2 158.6	54.6 52.9 57.4 98.1 108.6		14 808 653 946 1832 1600	11 1006 916 1077 1565 1542	21 692 860 1069 1106 1841
TA 98	-S9	0. 50. 160. 500. 1600. 5000.	15.3 201.7 320.3 663.0 1188.3 1956.0	0.6 23.0 18.8 169.0 200.7 213.3	13.2 20.9 43.3 77.7 127.8	INCOMPLETE	15 227 311 533 1068 2196	16 182 308 602 1077 1884	15 196 342 854 1420 1788
WP2uvrA	+S9	0. 50. 160. 500. 1600. 5000.	21.7 29.3 24.3 22.0 23.0 24.3	2.3 1.5 6.0 2.0 4.6 6.7	1.4 1.1 1.0 1.1 1.1		19 28 18 24 19 32	23 29 30 20 28 21	23 31 25 22 22 20
WP2uvrA	-S9	0. 50. 160. 500. 1600. 5000.	17.0 25.0 25.7 22.7 21.0 20.0	2.0 6.2 0.6 5.9 2.6 3.5	1.5 1.5 1.3 1.2 1.2		19 18 26 27 22 24	17 27 25 16 23 18	15 30 26 25 18 18

 

Historical control data

TA 100		With S9-mix		Without S9-mix
	Solvent control (DMSO)	Negative control	Positive control	Solvent control (DMSO)	Negative control	Positive control
Mean SD Range	134.2 25.7 73.0 – 204.0	141.2 26.3 79.0 – 209.7	1462.1 519.5 287.3 – 2893.7	123.8 25.6 73.0 – 186.7	137.4 28.8 81.0 – 238.0	597.0 425.9 296.3 – 2946.0
n	91.0	121.0	121.0	94.0	121.0	121.0

 

TA 1535		With S9-mix		Without S9-mix
	Solvent control (DMSO)	Negative control	Positive control	Solvent control (DMSO)	Negative control	Positive control
Mean SD Range	9.4 2.1 5.7 – 16.7	10.0 4.2 5.3 – 17.0	202.1 71.4 80.7 – 559.0	9.4 2.6 4.7 – 23.3	9.8 2.3 4.6 – 22.7	454.6 423.1 154.0 – 2701.0
n	89.0	118.0	117.0	89.0	118.0	118.0

 

TA 1537		With S9-mix		Without S9-mix
	Solvent control (DMSO)	Negative control	Positive control	Solvent control (DMSO)	Negative control	Positive control
Mean SD Range	7.0 2.4 2.3 – 14.0	7.0 2.5 3.0 – 17.7	256.1 183.4 60.0 – 1784.3	6.9 2.5 3.0 – 14.0	7.1 2.5 2.0 – 14.7	152.3 76.1 62.3 – 636.3
n	86.0	114.0	114.0	85.0	114.0	114.0

 

TA 98		With S9-mix		Without S9-mix
	Solvent control (DMSO)	Negative control	Positive control	Solvent control (DMSO)	Negative control	Positive control
Mean SD Range	23.0 4.8 11.7 – 38.3	24.3 5.6 15.3 – 44.7	1418.2 567.8 149.0 – 2511.0	20.0 4.9 8.3 – 40.3	21.0 5.7 13.0 – 40.0	412.4 167.1 120.7 – 1730.3
n	82.0	114.0	114.0	81.0	114.0	114.0

 

WP2uvrA		With S9-mix		Without S9-mix
	Solvent control (DMSO)	Negative control	Positive control	Solvent control (DMSO)	Negative control	Positive control
Mean SD Range	28.5 9.3 15.0 – 48.0	30.1 6.9 14.7 – 41.0	189.5 77.4 79.7 – 324.3	28.8 8.1 10.7 – 42.7	28.7 7.3 16.3 – 49.3	174.8 59.6 73.7 – 276.3
n	15.0	24.0	25.0	15.0	24.0	25.0

 

SD = standard deviation

n = number of experiment

Applicant's summary and conclusion

Conclusions:

The results lead to the conclusion that Resolln Blau BBLS Presskuchen trocken is mutagenic in the bacterial test systems with and without an exogenous metabolizing system.

Executive summary:

Resolin Blau BBLS Presskuchen trocken was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA.

 

A mutagenicity study (plate incorporation test) was conducted, in the absence and in the presence of a metabolizing system derived from a rat liver homogenate. For the study, the compound was suspended in DMSO, and each bacterial strain was exposed to 5 dose levels. Concentrations ranged from 50 to 5000 μg/plate.

 

Control plates without mutagen showed that the number of spontaneous revertant colonies was within the laboratory's historical control range. All the positive control compounds showed the expected increase in the number of revertant colonies.

 

Toxicity: In the mutagenicity experiment toxicity was not observed with metabolic activation. In the absence of metabolic activation the test compound proved to be toxic to all salmonella bacterial strains at a concentration of 5000 μg/plate.

 

Mutagenicity: In the absence and in the presence of the metabolic activation system Resolin Blau BBLS Presskuchen trocken gave a dose-dependent increase in the number of revertant colonies with the salmonella bacterial strains.

 

Summarizing, it can be stated that Resolin Blau BBLS Presskuchen trocken is mutagenic in the bacterial test systems in the absence and in the presence of exogenous metabolic activation.