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EC number: 944-073-5 | CAS number: -
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
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:
- 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 016
- Report date:
- 2017
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- Published in O.J. L 142 (2008)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Reaction products of 1,3-dihydroxybenzene, 4-amino-5-hydroxynaphthalene-2,7-disulphonic acid, 4-methoxy-1-aminobenzene, 2-(4-aminoanilino)-5-nitrobenzenesulphonic acid, sodium salt
- EC Number:
- 602-814-7
- Cas Number:
- 12269-90-8
- IUPAC Name:
- Reaction products of 1,3-dihydroxybenzene, 4-amino-5-hydroxynaphthalene-2,7-disulphonic acid, 4-methoxy-1-aminobenzene, 2-(4-aminoanilino)-5-nitrobenzenesulphonic acid, sodium salt
- Test material form:
- solid: particulate/powder
- Details on test material:
- Batch No.: 7012/2007
Appearance: brown powder
Composition of test substance:
Main component:
2-(4-aminoanilino)-5-nitrobenzenesulphonic acid 5.5 area %
4-[[2,4-dihydroxy-3,5-bis[[4-(4-nitro-2-sulphonatophenyl)amino]phenyl]azo]phenyl]azo]-5-
hydroxynaphtalen-2,7-disulphonate, sodium salts 13.9 area %
4-[[2,4-dihydroxy-3-[(4-methoxyphenyl)azo]-5-[[4-[(4-nitro-2-sulphonatophenyl)amino]phenyl]azo]phenyl]azo]-
5-hydroxynaphthalene-2,7-disulphonate, sodium salts 42.4 area %
4-[[2,4-dihydroxy-3,5-bis[(4-methoxyphenyl)azo]phenyl]azo]-5-
hydroxynaphthalene-2,7-disulphonate, sodium salts 18.2 area %
1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Test substance name: Acid Brown 235
- Other name: Korostan Brown DGR
- Source and lot/batch No. of test material: 7012/2007
- Expiration date of the lot/batch: May 2018
- Appearance: dark brown powder
Method
- Target gene:
- gene for histidine or tryptophan synthesis
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction of rat liver homogenate
- Test concentrations with justification for top dose:
- 50, 150, 500, 1500 and 5000 µg per plate
- Vehicle / solvent:
- water for injection
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- sodium azide
- other: 4-nitro-2-phenylenediamine (CAS: 99-56-9); 2-aminofluorene (CAS: 153-78-6); 2-aminoanthracene (CAS: 613-13-8); N-methyl-N´-nitro-N-nitrosoguanidine (CAS: 70-25-7);
- Details on test system and experimental conditions:
- CHEMICALS AND MEDIA
Solvent:
- water for injection
metabolic activator:
- S9: prepared in-house
Positive controls:
- sodium azide (AS) (CAS: 26647-22-8)
- 4-nitro-2-phenylenediamine (NPD) (CAS: 99-56-9)
- 2-aminofluorene (2-AF) (CAS: 153-78-6)
- 2-aminoanthracene (2-AA) (CAS: 613-13-8)
- N-methyl-N´-nitro-N-nitrosoguanidine (MNNG) (CAS: 70-25-7)
- 9-aminoacridine hydrochloride monohydrate (9-AAc) (CAS: 52417-22-8)
Media:
- Nutrient Broth for microbiology
- Nutrient agar
- Agar-agar
TEST SYSTEM
Bacterial strains:
The bacterial tester strains Salmonella typhimurium TA 1535 (CCM 3814, lot. No. 2101200916917), TA 98 (CCM 3811, lot No. 01022001220053), TA 100 (CCM 3812, lot No. 0102201220054) and TA 1537 (CCM 3815, lot No. 2101200916918) as well as Escherichia coli WP2 uvrA (CCM 4751, lot No. 2104200512732), - were obtained from Czech Collection of Microorganisms (CCM) of Masaryk University, Brno. Strains TA 98 and TA 1537 detect frame shift mutations, strains TA 100 and TA 1535 serve to detection of base-pair substitution mutations, and strain E.coli WP2 uvrA detects cross-linking mutagens.
Genotypes of strains:
Genotypes of each strain were controlled (plasmid pKM 101 – ampicillin resistance, uvr mutation, rfa mutation, his/trp mutation – spontaneous reversions).
Preparation and using of S9:
The metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors. The liver homogenate was prepared from Wistar male rats weighing approximately 200 g, previously induced with Delor 106 (mixture of PCBs). Delor 106 was diluted with olive oil to a concentration of 200 mg/mL, and each rat was administered a single injection of 500 mg/kg 5 days before S9 preparation. The S9 was prepared according to the methods described by Maron and Ames (1983). The liver was removed from each animal and washed in ice cold 0.15M KCl. The livers washed were mixed with another 0.15 M KCl (3 mL/g wet liver) homogenized in a grinder, and the tissue suspension was centrifuged for 10 min at 9000 g. Aliquots of the supernatant (S9) were stored in plastic tubes using sterile technique at a temperature below –70°C. Cofactors (NADP and glucoso-6-phosphate) were dissolved in buffer.
Each plate in all experiments with metabolic activation contained 0.5 mL of buffer with NADP and glucoso-6-phosphate and 30 or 100 µL S9 (the concentration of S9 in the S9mix was 5.7 or 19%). In experiments without metabolic activation only buffer was added to the top agar.
Controls:
Each experiment included corresponding positive (reference mutagens) and negative controls (untreated control, solvent control). Untreated controls contain no solvent and negative controls contain 0.1 mL of water. All the control numbers were compared with historical ranges of mutant frequencies obtained in our laboratory. The actual numbers were in ranges of the historical numbers.
PLATE INCORPORATION TEST
Test procedure:
100 µL of the test substance of required concentration, 100 µL of 16-18 h culture of tester strain of density 10^8-10^9 CFU/mL, 0.5 mL relevant buffer and 30, 50 or 100 µL of S9 post mitochondrial fraction (in case of test with metabolic activation) were added to the 2 mL of molten top agar (with trace of histidine or tryptophan) kept in a test tube at 45±3°C. After shaking the mixture was poured into a minimal glucose agar plate.
Petri dishes were incubated of 48 - 72 h at 37±1°C, the number of revertant colonies on the plate was counted manually with exception of positive controls, which were counted by an AccuCount 1000. For an adequate estimate of variation, triplicate plating was used at each dose level. The toxicity test, which serves for finding of optimal concentrations for the mutagenicity test, was performed in strain TA 98 and two Petri dishes were used for every concentration.
Selection of doses/toxicity:
The test substance was dissolved in water for injection till the maximum recommended concentration 5000 μg per 0.1 mL. For toxicity experiment the highest concentration was diluted to the other 5 concentrations in 3 digit places interval. Although no particles could be observed in higher concentrations due to dark colour of solutions, no particles were observable in top agar and Petri dishes.
The concentration row was tested for toxicity in strain TA 100 without metabolic activation.
No toxicity or precipitation was observed in any dose. The concentration of 5000 µg per 0.1 mL-1 was then used as maximum in the first mutagenicity experiments. Further doses were diluted with factor approximately 2· √10. Mutagenicity occurred in some cases in higher doses so lower concentrations were omitted and generally higher concentrations were used in the second mutagenicity experiments. For increase of sensibility, the second experiments were performed with pre-incubation for 30 minutes at 37±1°C and shaking. Fresh solutions of the test substance were prepared before each experiment. Concentrations of the test substance solution were dosed in the volume of 0.1 mL per plate. - Evaluation criteria:
- EVALUATION OF RESULTS
The main criterion for evaluation of results was modified two-fold increase rule, which is compatible with the application of statistical methods. After this rule the result is positive, if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc is reached.
An increase is considered as ”biologically relevant“:
- if the number of reversions is at least twice as high as that in the solvent control for the strains having spontaneous reversion >10;
- if the number of reversions is at least three times as high as that in the solvent control for the strains having spontaneous reversion ≤10;
A test substance producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system. According to OECD TG 471, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary. - Statistics:
- 1. Maron D. M., Ames B. N., Revised methods for the Salmonella mutagenicity test, Mutation Research, 113, p. 173 - 215 (1983);
2. Dunkel V. C., Chu K.C., Evaluation of methods for analysis of microbial mutagenicity assays, The Predictive Value of Short-Term Screening Tests in Carcinogenicity Evaluation, Elsevier North-Holland Biomedical Press, p. 231 - 417 (1980);
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
Any other information on results incl. tables
Table 1: The effect of the test substance
Doses µg/plate |
S9 µL |
revertants per plate |
mean ±sd |
Rt/Rc |
S9 µL |
revertants per plate |
mean ±sd |
Rt/Rc |
||||||||||||
Escherichia coli WP2 uvrA |
||||||||||||||||||||
|
experiment I |
|||||||||||||||||||
Sp.rev. |
0 |
35 |
39 |
31 |
35± 3 |
- |
100 |
33 |
45 |
29 |
36± 7 |
- |
||||||||
water |
0 |
34 |
28 |
35 |
32± 3 |
- |
100 |
34 |
35 |
32 |
34± 1 |
- |
||||||||
50 |
0 |
34 |
32 |
35 |
34± 1 |
1.0 |
100 |
28 |
28 |
39 |
32± 5 |
0.9 |
||||||||
150 |
0 |
30 |
37 |
36 |
34± 3 |
1.1 |
100 |
35 |
32 |
33 |
33± 1 |
1.0 |
||||||||
500 |
0 |
33 |
33 |
41 |
36± 4 |
1.1 |
100 |
35 |
33 |
37 |
35± 2 |
1.0 |
||||||||
1500 |
0 |
68 |
62 |
50 |
60± 7 |
1.9 |
100 |
39 |
26 |
44 |
36± 8 |
1.1 |
||||||||
5000 |
0 |
114 |
140 |
131 |
128±11 |
4.0 |
100 |
96 |
105 |
71 |
91±14 |
2.7 |
||||||||
MNNG/2-AA |
0 |
1038 |
945 |
NT |
992±47 |
30.7 |
100 |
147 |
153 |
NT |
150± 3 |
4.5 |
||||||||
|
experiment II |
|||||||||||||||||||
Sp. rev. |
0 |
40 |
38 |
32 |
37± 3 |
- |
100 |
36 |
46 |
49 |
44± 6 |
- |
||||||||
water |
0 |
38 |
44 |
32 |
38± 5 |
- |
100 |
43 |
42 |
39 |
41± 2 |
- |
||||||||
1000 |
0 |
88 |
58 |
67 |
71±13 |
1.9 |
100 |
40 |
42 |
36 |
39± 2 |
1.0 |
||||||||
2000 |
0 |
85 |
90 |
104 |
93± 8 |
2.4 |
100 |
47 |
52 |
52 |
50± 2 |
1.2 |
||||||||
3000 |
0 |
133 |
113 |
100 |
115±14 |
3.0 |
100 |
64 |
66 |
47 |
59± 9 |
1.4 |
||||||||
4000 |
0 |
126 |
139 |
169 |
145±18 |
3.8 |
100 |
99 |
81 |
114 |
98±13 |
2.4 |
||||||||
5000 |
0 |
172 |
176 |
170 |
173± 2 |
4.5 |
100 |
126 |
133 |
138 |
132± 5 |
3.2 |
||||||||
MNNG/2-AA |
0 |
1028 |
1022 |
NT |
1025±3 |
27.0 |
100 |
207 |
196 |
NT |
202± 6 |
4.9 |
||||||||
Salmonella typhimurium TA 1537 |
||||||||||||||||||||
|
experiment I |
|||||||||||||||||||
Sp.rev. |
0 |
12 |
7 |
7 |
9± 2 |
- |
30 |
15 |
16 |
11 |
14± 2 |
- |
||||||||
water |
0 |
9 |
11 |
11 |
10± 1 |
- |
30 |
16 |
8 |
14 |
13± 3 |
- |
||||||||
50 |
0 |
10 |
18 |
10 |
13± 4 |
1.2 |
30 |
19 |
15 |
18 |
17± 2 |
1.4 |
||||||||
150 |
0 |
10 |
9 |
18 |
12± 4 |
1.2 |
30 |
16 |
15 |
21 |
17± 3 |
1.4 |
||||||||
500 |
0 |
16 |
11 |
11 |
13± 2 |
1.2 |
30 |
18 |
14 |
15 |
16± 2 |
1.2 |
||||||||
1500 |
0 |
23 |
25 |
21 |
23± 2 |
2.2 |
30 |
28 |
25 |
17 |
23± 5 |
1.8 |
||||||||
5000 |
0 |
71 |
52 |
60 |
61± 8 |
5.9 |
30 |
37 |
45 |
44 |
42± 4 |
3.3 |
||||||||
9-AAc/2-AA |
0 |
980 |
1109 |
NT |
1045±65 |
101.1 |
20 |
100 |
117 |
NT |
109± 9 |
8.6 |
||||||||
|
experiment II |
|||||||||||||||||||
Sp. rev. |
0 |
8 |
13 |
8 |
10± 2 |
- |
30 |
11 |
18 |
0 |
15± 4 |
- |
||||||||
water |
0 |
10 |
11 |
12 |
11± 1 |
- |
30 |
10 |
18 |
14 |
14± 3 |
- |
||||||||
1000 |
0 |
18 |
23 |
18 |
20± 2 |
1.8 |
30 |
12 |
17 |
17 |
15± 2 |
1.1 |
||||||||
2000 |
0 |
29 |
29 |
29 |
29± 0 |
2.6 |
30 |
21 |
19 |
22 |
21± 1 |
1.5 |
||||||||
3000 |
0 |
36 |
42 |
28 |
35± 6 |
3.2 |
30 |
30 |
41 |
35 |
35± 4 |
2.5 |
||||||||
4000 |
0 |
48 |
44 |
40 |
44± 3 |
4.0 |
30 |
49 |
50 |
47 |
49± 1 |
3.5 |
||||||||
5000 |
0 |
39 |
41 |
55 |
45± 7 |
4.1 |
30 |
66 |
43 |
47 |
52± 10 |
3.7 |
||||||||
9-AAc/2-AA |
0 |
1291 |
1314 |
NT |
1303±12 |
118.4 |
20 |
201 |
201 |
NT |
201±0 |
14.4 |
||||||||
Salmonella typhimurium TA 100 |
||||||||||||||||||||
|
experiment I |
|||||||||||||||||||
Sp.rev. |
0 |
85 |
89 |
111 |
95±11 |
- |
30 |
104 |
105 |
112 |
107± 4 |
- |
||||||||
water |
0 |
89 |
121 |
102 |
104±13 |
- |
30 |
111 |
96 |
118 |
108± 9 |
- |
||||||||
50 |
0 |
112 |
111 |
109 |
111± 1 |
1.1 |
30 |
108 |
c |
107 |
108± 1 |
1.0 |
||||||||
150 |
0 |
98 |
87 |
95 |
93± 5 |
0.9 |
30 |
88 |
96 |
111 |
98±10 |
0.9 |
||||||||
500 |
0 |
101 |
95 |
106 |
101± 4 |
1.0 |
30 |
105 |
95 |
127 |
109±13 |
1.0 |
||||||||
1500 |
0 |
114 |
124 |
102 |
113± 9 |
1.1 |
30 |
127 |
128 |
121 |
125± 3 |
1.2 |
||||||||
5000 |
0 |
140 |
128 |
158 |
142±12 |
1.4 |
30 |
135 |
131 |
184 |
150±24 |
1.4 |
||||||||
AS/2-AF |
0 |
481 |
511 |
NT |
496±15 |
4.8
|
20 |
1315 |
1328 |
NT |
1322± 7 |
12.2 |
||||||||
|
experiment II |
|||||||||||||||||||
Sp. rev. |
0 |
83 |
78 |
75 |
79± 3 |
- |
30 |
84 |
108 |
91 |
94±10 |
- |
||||||||
water |
0 |
84 |
106 |
82 |
91±11 |
- |
30 |
97 |
89 |
105 |
97± 7 |
- |
||||||||
1000 |
0 |
105 |
113 |
127 |
115± 9 |
1.3 |
30 |
90 |
96 |
86 |
91± 4 |
0.9 |
||||||||
2000 |
0 |
139 |
135 |
132 |
135± 3 |
1.5 |
30 |
110 |
107 |
106 |
108± 2 |
1.1 |
||||||||
3000 |
0 |
138 |
161 |
136 |
145±11 |
1.6 |
30 |
110 |
131 |
120 |
120± 9 |
1.2 |
||||||||
4000 |
0 |
129 |
134 |
140 |
134± 4 |
1.5 |
30 |
132 |
122 |
140 |
131± 7 |
1.4 |
||||||||
5000 |
0 |
180 |
183 |
171 |
178± 5 |
2.0 |
30 |
134 |
154 |
135 |
141± 9 |
1.5 |
||||||||
AS/2-AF |
0 |
492 |
492 |
NT |
492± 0 |
5.4 |
20 |
950 |
1034 |
NT |
992±42 |
10.2 |
||||||||
Salmonella typhimurium TA 1535 |
||||||||||||||||||||
|
experiment I |
|||||||||||||||||||
Sp.rev. |
0 |
21 |
17 |
25 |
21± 3 |
- |
30 |
18 |
23 |
16 |
19± 3 |
- |
||||||||
water |
0 |
19 |
20 |
25 |
21± 3 |
- |
30 |
12 |
19 |
12 |
14± 3 |
- |
||||||||
50 |
0 |
22 |
27 |
29 |
26± 3 |
1.2 |
30 |
14 |
15 |
12 |
14± 1 |
1.0 |
||||||||
150 |
0 |
20 |
24 |
21 |
22± 2 |
1.0 |
30 |
16 |
16 |
12 |
15± 2 |
1.0 |
||||||||
500 |
0 |
15 |
28 |
27 |
23± 6 |
1.1 |
30 |
18 |
12 |
11 |
14± 3 |
1.0 |
||||||||
1500 |
0 |
19 |
24 |
24 |
22± 2 |
1.0 |
30 |
18 |
15 |
10 |
14± 3 |
1.0 |
||||||||
5000 |
0 |
27 |
20 |
23 |
23± 3 |
1.1 |
30 |
25 |
14 |
24 |
21± 5 |
1.5 |
||||||||
AS/2-AA |
0 |
410 |
419 |
NT |
415± 5 |
19.4 |
20 |
184 |
150 |
NT |
167±17 |
11.7 |
||||||||
|
experiment II |
|||||||||||||||||||
Sp. rev. |
0 |
28 |
18 |
18 |
21± 5 |
- |
30 |
17 |
22 |
19 |
19± 2 |
- |
||||||||
water |
0 |
23 |
21 |
24 |
23± 1 |
- |
30 |
20 |
15 |
24 |
20± 4 |
- |
||||||||
1000 |
0 |
21 |
17 |
16 |
18± 2 |
0.8 |
30 |
15 |
24 |
19 |
19± 4 |
1.0 |
||||||||
2000 |
0 |
18 |
24 |
19 |
20± 3 |
0.9 |
30 |
k |
15 |
21 |
18± 3 |
0.9 |
||||||||
3000 |
0 |
21 |
27 |
20 |
23± 3 |
1.0 |
30 |
15 |
18 |
24 |
19± 4 |
1.0 |
||||||||
4000 |
0 |
25 |
20 |
22 |
22± 2 |
1.0 |
30 |
25 |
19 |
17 |
20± 3 |
1.0 |
||||||||
5000 |
0 |
24 |
24 |
19 |
22± 2 |
1.0 |
30 |
21 |
25 |
32 |
26± 5 |
1.3 |
||||||||
AS/2-AA |
0 |
414 |
444 |
NT |
429±15 |
18.9 |
20 |
191 |
196 |
NT |
194±3 |
9.8 |
||||||||
Salmonella typhimurium TA 98 |
||||||||||||||||||||
|
experiment I |
|||||||||||||||||||
Sp.rev. |
0 |
31 |
25 |
31 |
29± 3 |
- |
30 |
30 |
39 |
39 |
36± 4 |
- |
||||||||
water |
0 |
25 |
30 |
25 |
27± 2 |
- |
30 |
40 |
35 |
36 |
37± 2 |
- |
||||||||
50 |
0 |
26 |
29 |
29 |
28± 1 |
1.1 |
30 |
35 |
40 |
25 |
33± 6 |
0.9 |
||||||||
150 |
0 |
26 |
22 |
23 |
24± 2 |
0.9 |
30 |
38 |
40 |
29 |
36± 5 |
1.0 |
||||||||
500 |
0 |
27 |
29 |
30 |
29± 1 |
1.1 |
30 |
38 |
33 |
26 |
32± 5 |
0.9 |
||||||||
1500 |
0 |
24 |
27 |
30 |
27± 2 |
1.0 |
30 |
32 |
25 |
23 |
27± 4 |
0.7 |
||||||||
5000 |
0 |
31 |
36 |
30 |
32± 3 |
1.2 |
30 |
39 |
36 |
34 |
36± 2 |
1.0 |
||||||||
NPD/2-AF |
0 |
664 |
817 |
NT |
741±77 |
27.8 |
20 |
1796 |
1871 |
NT |
1834±38 |
49.6 |
||||||||
|
experiment II |
|||||||||||||||||||
Sp.rev. |
0 |
32 |
31 |
24 |
29± 4 |
- |
30 |
35 |
48 |
38 |
40± 6 |
- |
||||||||
water |
0 |
30 |
33 |
34 |
32± 2 |
- |
30 |
38 |
44 |
41 |
41± 2 |
- |
||||||||
1000 |
0 |
38 |
46 |
32 |
39± 6 |
1.2 |
30 |
41 |
39 |
34 |
38± 3 |
0.9 |
||||||||
2000 |
0 |
35 |
31 |
34 |
33± 2 |
1.0 |
30 |
37 |
33 |
28 |
33± 4 |
0.8 |
||||||||
3000 |
0 |
43 |
38 |
37 |
39± 3 |
1.2 |
30 |
42 |
37 |
36 |
38± 3 |
0.9 |
||||||||
4000 |
0 |
56 |
115 |
36 |
69±34 |
2.1 |
30 |
36 |
34 |
38 |
36± 2 |
0.9 |
||||||||
5000 |
0 |
55 |
75 |
77 |
69±10 |
2.1 |
30 |
56 |
56 |
43 |
52± 6 |
1.3 |
||||||||
NPD/2-AF |
0 |
615 |
658 |
NT |
637±22 |
19.7 |
20 |
1571 |
1578 |
NT |
1575± 4 |
38.4 |
- S9 without metabolic activation
+ S9 with metabolic activation
CFU colony forming units, number of live bacteria in suspension
sd standard deviation
Rt/Rc ratio of number of revertants at tested dose to number of revertants in negative control
S9 amount of supernatant of rat liver homogenate per plate
Sp.rev. spontaneous reversion (untreated control)
AS sodium azide
NPD 4-nitro-2-phenylenediamine
2-AF 2-aminofluorene
2-AA 2-aminoanthracene
9-AAc 9-aminoacridine hydrochloride monohydrate
MNNG N-methyl-N´-nitro-N-nitrosoguanidine
NT not tested
c contamination
Applicant's summary and conclusion
- Conclusions:
- Under the test conditions, the test substance, Acid Brown 235, was mutagenic for the Salmonella typhimurium TA 1537, Salmonella typhimurium TA 100 and Escherichia coli WP2uvrA in experiments with as well as without metabolic activation.
The test substance was non-mutagenic for Salmonella typhimurium TA 98 and TA 1535 in experiments with as well as without metabolic activation. - Executive summary:
The test substance, Acid Brown 235, was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The performed test was based on EU method B.13/14 Mutagenicity – Reverse mutation test using bacteria,which is analogous to the OECD Test Guideline No. 471.
Four indicator, Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537, and one indicator, Escherichia coliWP2 uvrA strain, were used. The test substance was diluted in water for injection and assayed in doses of 50 - 5000 mg per plate, which were applied to plates in volume of 0.1 mL. The first mutagenicity experiments were performed without and with metabolic activation using a supernatant of rat liver (30 μL or 100 μL per plate) and a mixture of cofactorsby the plate in corporation test with a dose range of 50 – 5000mg per plate. Some signs of a positive response were observed in some tester strains, so, to increase the sensitivity of the assay, the second mutagenicity experiments were performed with pre-incubation and concentrations were shifted towards expected mutagenic doses to obtain a dose-response.
The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. Mean revertant colony counts for the vehicle controls were within the current historical control range for the laboratory.
Under the test conditions, the test substance, Acid Brown235, was mutagenic for the Salmonella typhimurium TA 1537, Salmonella typhimurium TA 100 and Escherichia coli WP2uvrA in experiments with as well as without metabolic activation.
The test substance was non-mutagenic for Salmonella typhimurium TA 98 and TA 1535 in experiments with as well as without metabolic activation.
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