2-[[4-(diethylamino)-2-methylphenyl]azo]-5-nitrobenzene-1,3-dicarbonitrile

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

11 November 1987 to 4 December 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-[[4-(diethylamino)-2-methylphenyl]azo]-5-nitrobenzene-1,3-dicarbonitrile

EC Number:

284-395-7

EC Name:

2-[[4-(diethylamino)-2-methylphenyl]azo]-5-nitrobenzene-1,3-dicarbonitrile

Cas Number:

84870-65-5

Molecular formula:

C19H18N6O2

IUPAC Name:

2-[[4-(diethylamino)-2-methylphenyl]azo]-5-nitrobenzene-1,3-dicarbonitrile

Test material form:

solid

Method

Target gene:

Histidine

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 specified

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material to the tester organisms. In the preliminary toxicity study, Dispersionsblau F-60 768 exhibited no toxicity to any of the strains of Salmonella at the maximum dose of 5000 ug/well.
In the main study five concentrations of the test material were assayed in triplicate against each tester strain, using the direct plate incorporation method in accordance with the standards for mutagenicity tests.
0, 8, 40, 200, 1000, 5000 μg/plate

Vehicle / solvent:

Dispersionsblau F-60 768 was accurately weighed and dissolved in dimethyl sulphoxide and appropriate dilutions made.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

other: N-Methyl-N'-Nitro-N-Nitrosoguanidine (MNNG); 4 NItro-O-phenyldiamine (4NOPD); 2-Aminoanthracene (2-AA)

Details on test system and experimental conditions:

Test Procedure
Preliminary Toxicity Study
In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material to the tester organisms. 0.1 ml of bacterial suspension was added to 4 ml of molten, trace histidine supplemented media (histidine/biotin & top agar) and overlayed onto sterile plates of Vogel Bonner agar (minimal agar- 25 ml/plate). When the agar had set, wells were made on each plate and 0.1 ml aliquots of concentrations ranging from 80 μg/ml to 50,000 μg/ml of test material were added to each well. These plates were incubated at 37 °C for at least 24 hours after which time the toxicity of the test material was assessed by measuring the zones of inhibition around each well.
Dimethyl sulphoxide, which was used as a solvent diluent for the test material in this assay, was also added to each plate as a control.

Mutation Study
Experiment 1
Five concentrations of the test material were assayed in triplicate against each tester strain, using the direct plate incorporation method in accordance with the standards for mutagenicity tests using micro-organisms.

Test Material and Negative Controls
0.1 ml of the appropriately diluted test material or negative control solution was placed in sets of sterile test tubes containing 2.0 ml of molten, trace histidine supplemented, top agar at 45 °C. These sets comprised of two test tubes for each bacterial tester strain. A 0.1 ml aliquot of one of the bacterial suspensions was also added to each of the two test tubes. Into one of the test tubes was placed 0.5 ml of the S-9 liver microsome mix; in the other tube 0.5 ml of pH 7.4 buffer was added. This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material.

Positive Controls
Without Activation
0.1 ml of one of the appropriate positive control solutions (MNNG, 9AA, or 4NOPD) was added to a test tube containing 2.0 ml of molten, trace histidine supplemented, top agar. 0.1 ml of bacterial suspension and 0.5 ml of pH 7.4 buffer was also added to the agar. This procedure was then repeated, in triplicate, for each of the positive controls and each of the bacterial strains.
With Activation
0.1 ml of 2AA solution was added to a test tube containing 2.0 ml of molten, trace histidine supplemented, top agar. 0.1 ml of one of the test bacterial suspensions and 0.5 ml of S-9 mix were also added to the agar. The procedure was then repeated, in triplicate, for each tester strain.
The contents of each test tube were equally distributed onto the surface of Vogel-Bonner agar plates (one tube per plate). These plates were incubated at 37 °C for at least 48 hours and the number of revertant colonies counted.

Experiment 2
The complete experiment was repeated using fresh bacterial cultures, test material and control solutions.

Tester Strains
The strains used in this assay were all mutants derived from Salmonella typhimurium LT2 and were those recommended for general screening.
TA1535: sensitive to agents inducing base-pair substitution
TA100: sensitive to agents inducing base-pair substitution
TA1537: sensitive to agents inducing frame-shift mutations
TA1538: sensitive to agents inducing frame-shift mutations
TA98: sensitive to agents inducing frame-shift mutations

These strains were obtained from the British Industrial Biological Research Association on 14th August 1987 and were stored at 4 °C on nutrient agar slopes. Prior to being used, characterisation checks were carried out to determine the amino-acid requirement, presence of rfa and R factors and the spontaneous reversion rate.
In this assay, overnight sub-cultures of the master slopes were prepared in nutrient broth (Oxoid Limited) and incubated at 37 °C for approximately 18 hours. These overnight cultures yielded approximately 107 - 109 bacteria per ml.

Microsomal Enzyme Fraction
Lot No. Aro. S-9/08/10, prepared on 8 October 1987 was obtained from the British Industrial Biological Research Association on 8 October 1987. For the second experiment Lot. No. Aro. S-9/17/11, prepared on 17 November 1987 was obtained from B.I.B.R.A. on 19 November 1987. They were prepared from the livers of male Sprague-Dawley rats weighing 150 - 200g. These had received a single i.p. injection of Aroclor 1254 at 500 mg/kg, 5 days before S-9 preparation.
The S-9 was stored at -180 °C in a Statebourne liquid N2 freezer, model SXR 34.

Rationale for test conditions:

The study was conducted according to Laboratory Protocol and was designed to assess the mutagenic potential of the test material using a bacterial/microsome test system. The study was based on the in vitro technique described by Ames and his co-workers and Garner et al in which mutagenic activity is assessed by exposing histidine auxotrophs of Salmonella typhimurium to various concentrations of the test material.

Evaluation criteria:

For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate (of at least twice the spontaneous reversion rate) in one or more strains of bacteria in the presence and/or absence of the S-9 microsomal enzymes in both experiments. To be considered negative the number of induced revertants compared to spontaneous revertants should be less than twofold at all dose levels employed, the intervals of which should be between 2- and 5-fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5 mg/plate.

Statistics:

Statistical methods not referenced in the study report.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Study
Dispersionsblau F 60 768 exhibited no toxicity to any of the strains of Salmonella at the maximum dose of 5000 μg/well.

Mutation Study
The overnight culture of each strain was found to be in the required range of 107 to 109 bacteria per ml and the spontaneous reversion rate for each was found to be within the expected range.
A marked precipitate of Dispersionsblau F-60 768 was seen at 5000 μg/plate in both experiments and intermittently at 2500 ug/plate in the second experiment. In some cases the precipitate prevented accurate recording of the numbers of revertant colonies, especially where the revertant colonies were small.
Dispersionsblau F-60 768 induced a highly significant dose-related increase in the number of bacterial revertant colonies in all the Salmonella strains both with and without metabolic activation. An increase was noted between a range of 8 - 5000 μg/plate with the maximum response recorded at a dose level of 2500 μg/plate. In all cases the response was greater in the presence of metabolic activation than in its absence. Also, the response seen in the bacterial strains that detect frameshift mutations (TA1537, TA1538 and TA98) was moderately stronger than that seen in the base-substitution strains (TA1535 and TA100).

The positive control substances all produced marked increases in the number of revertant colonies and the activity of the S-9 fraction was found to be satisfactory.

Any other information on results incl. tables

VIABILITY AND SPONTANEOUS REVERSION TESTS

EXPERIMENT 1

Strain of Salmonella typhimurium	Bacteria per 1 ml of overnight nutrient broth	Total counts on trace histidine supplemented top agar
TA1535   TA 1537   TA 1538   TA98   TA100	15 x 10^7   1 x 10^7   8 x 10^7   11 x 10^7   12 x 10^7	22   8   20   25   150	36   9   28   20   138	31   11   30   18   153

EXPERIMENT 2

Strain of Salmonella typhimurium	Bacteria per 1 ml of overnight nutrient broth	Total counts on trace histidine supplemented top agar
TA1535   TA 1537   TA 1538   TA98   TA100	15 x 10^7   34 x 10^7   5 x 10^7   127 x 10^7   52 x 10^7	19   4   15   22   144	12   8   18   28   129	15   9   14   17   97

 

 

EXPERIMENT 1 – MUTATION STUDY INDIVIDUAL PLATE COUNTS

FOR TEST MATERIAL AND NEGATIVE CONTROLS

Strain of Salmonella typhimurium	Test Material	Conc. μg/plate	Metabolic activation	Mean No. of revertants	Individual plate count
TA1535	DISPERSIONSBLAU F-60 768	5000 1000 200 40 8 0	- - - - - -	36 29 23 20 18 12	40P 30 26 20 17 12	35P 31 21 22 19 12	32P 27 23 19 ° 11
		5000 1000 200 40 8 0	+ + + + + +	135 62 35 15 13 15	144P 61 27 16 17 13	124P 66 47 11 9 15	137P 58 32 17 3 16
TA1537	DISPERSIONSBLAU F-60 768	5000 1000 200 40 8 0	- - - - - -	34 27 26 20 11 10	37P 22 18 13 15 13	30P 31 33 24 11 9	35P 29 27 23 8 8
		5000 1000 200 4 8 0	+ + + + + +	471 410 141 31 15 15	430P 360 136 29 11 15	492P 416 126 29 18 12	492P 454 160 35 16 198
TA1538	DISPERSIONSBLAU F-60 768	5000 1000 200 40 8 0	- - - - - -	591 496 272 167 76 9	578P 500 295 162 80 10	636P 500 262 170 67 7	558P 488 260 168 82 11
		5000 1000 200 40 8 0	+ + + + + +	1084 1868 1485 191 45 15	1169P 1967 1376 188 52 15	1412P 1880 1640 201 39 9	671P 1758 1440 184 43 22
TA98	DISPERSIONSBLAU F-60 768	5000 1000 200 40 8 0	- - - - - -	863 847 447 240 113 19	890P 864 432 246 113 22	940P 798 476 274 110 16	760P 878 432 200 115 19
		5000 1000 200 40 8 0	+ + + + + +	1567 1931 1387 189 58 22	1680P 1888 1520 156 56 22	1620P 1930 1200 182 66 23	1400P 1974 1440 230 52 20
TA100	DISPERSIONSBLAU F-60 768	5000 1000 200 40 8 0	- - - - - -	567 394 319 195 156 93	660P 381 306 175 161 77	493P 391 336 205 146 92	548P 411 315 205 161 109
		5000 1000 200 40 8 0	+ + + + + +	1451 1601 761 236 141 133	1578P 1622 751 236 148 152	1369P 1666 764 243 126 109	1405P 1516 768 230 148 138

° = plate unscorable           P = precipitate

 

EXPERIMENT 2 – MUTATION STUDY INDIVIDUAL PLATE COUNTS

FOR TEST MATERIAL AND NEGATIVE CONTROLS

Strain of Salmonella typhimurium	Test Material	Conc. μg/plate	Metabolic activation	Mean No. of revertants	Individual plate count
TA1535	DISPERSIONSBLAU F-60 768	5000 2500 1250 625 312.5 0	- - - - - -	38 27 30 32 26 20	31P 37 30 36 30 27	37P 21 41 31 16 22	46P 24 18 28 33 12
		5000 2500 1250 625 312.5 0	+ + + + + +	113 88 65 54 40 21	132P 84 56 49 43 21	91P 100 69 57 34 21	117P 80 70 57 44 20
TA1537	DISPERSIONSBLAU F-60 768	500 2500 1250 625 312.5 0	- - - - - -	65 41 21 27 21 8	82P 35P 28 22 17 9	66P 33P 17 32 25 9	48P 55P 17 26 20 7
		5000 2500 1250 625 312.5 0	+ + + + + +	609 641 458 266 135 11	686P 611 441 248 150 10	563P 624 495 278 126 13	577P 687 438 271 129 11
TA1538	DISPERSIONSBLAU F-60 768	5000 2500 1250 625 312.5 0	- - - - - -	- 638 341 290 268 17	°P 640P 358 272 286 21	°P 635P 309 301 239 16	°P 638P 355 298 278 13
		5000 2500 1250 625 312.5 0	+ + + + + +	1202 1624 1177 851 751 24	1100P 1706 1125 841 699 28	1011P 1577 1225 910 803 19	1495P 1588 1181 803 752 25
TA98	DISPERSIONSBLAU F-60 768	5000 2500 1250 625 312.5 0	- - - - - -	866 1009 700 541 431 16	842P 1069P 706 544 460 19	907P 939P 709 544 401 13	849P 1018P 684 535 431 15
		5000 2500 1250 625 312.5 0	+ + + + + +	2415 2637 2167 1400 1106 29	2775P 2640 2078 1576 1173 20	2465P 2652 2064 1481 1124 38	2004P 2620 2360 1144 1020 30
TA100	DISPERSIONSBLAU F-60 768	5000 2500 1250 625 312.5 0	- - - - - -	819 638 299 268 222 78	694P 518P 292 264 232 83	993P 819P 304 274 201 81	770P 578P 300 263 233 70
		5000 2500 1250 625 312.5 0	+ + + + + +	1362 1592 1083 835 566 126	1706P 1331 958 828 638 127	1298P 1984 1184 840 488 128	1082P 1461 1106 837 572 124

° = plate unscorable           P = precipitate

 

MUTABILITY TESTS WITH POSITIVE CONTROLS

Strain of Salmonella typhimurium	Test Material	Conc. μg/plate	Metabolic activation	Mean No. of revertants	Individual plate count
EXPERIMENT 1
TA1535 TA1537 TA1538 TA98 TA100 TA1535 TA1537 TA1538 TA98 TA100	MNNG 9AA 4NOPD 4NOPD MNNG 2AA 2AA 2AA 2AA 2AA	2 100 10 10 2 3.3 3.3 3.3 3.3 3.3	- - - - - + + + + +	737 1283 867 1223 1489 589 458 344 310 522	1009 1369 826 1200 1454 737 630 495 379 540	224 1190 781 1306 1458 532 383 388 277 506	979 1291 993 1162 1556 499 362 148 273 520
EXPERIMENT 2
TA1535 TA1537 TA1538 TA98 TA100 TA1535 TA1537 TA1538 TA98 TA100	MNNG 9AA 4NOPD 4NOPD MNNG 2AA 2AA 2AA 2AA 2AA	2 100 10 10 2 3.3 3.3 3.3 3.3 3.3	- - - - - + + + + +	774 681 759 715 887 137 188 268 1044 1666	781 626 693 758 888 125 187 267 1070 1712	758 705 746 693 923 142 194 285 1005 1629	782 711 839 694 849 144 182 252 1057 1657

 

Applicant's summary and conclusion

Conclusions:

The test material, Dispersionsblau F 60 768, was found to exhibit evidence of mutagenic activity under the conditions of this test. The substance is mutagenic in bacteria with and without a metabolic activation system.

Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 amd TA100 were treated with Dispersionsblau F-60 768 by the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system at 10% in standard co-factors. The dose range was determined in a preliminary toxicity assay and was 8 to 5000 μg/plate in the first experiment. The experiment was repeated on a separate day using different cultures of the bacterial strains and fresh chemical solutions. In this case the dose range of Dispersionsblau F-60 768 was 312.5 to 5000 μg/pJate.

 

All solvent (DMSO) control plates gave counts of revertant colonies within the normal range.

 

All positive control chemicals gave increases in revertants, both with and without the metabolising system, within expected ranges.

 

Dispersionsblau F-60 768 did not cause a reduction in the growth of the bacterial lawn at any dose level in any of the strains of Salmonella.

Dispersionsblau F-60 768 was, therefore, tested up to the maximum recommended dose of 5000 μg/plate.

 

A significant dose-related increase in the numbers of revertant colonies was recorded for all of the bacterial strains at doses ranging from 8 - 5000 μg/plate both with and without metabolic activation in both experiments. Dispersionsblau F-60 768 was found to exhibit evidence of marked mutagenic activity under the conditions of this test.