Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2006

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Salmonella/Mammalian-Microsome Mutagenicity Assay. The experimental materials, methods, and procedures are based on those described by Ames et al. (Ames, B. N., McCann, J., and Yamasaki, E. (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat. Res. 31, 347-364).
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
trisodium 6-[(1E)-2-(2,4-diaminophenyl)diazen-1-yl]-3-[(1E)-2-[4-({4-[(1Z)-2-{7-[(1E)-2-(2,4-diaminophenyl)diazen-1-yl]-1-hydroxy-3-sulfonatonaphthalen-2-yl}diazen-1-yl]-2-sulfonatophenyl}amino)phenyl]diazen-1-yl]-4-hydroxynaphthalene-2-sulfonate
EC Number:
939-382-7
Molecular formula:
not applicable
IUPAC Name:
trisodium 6-[(1E)-2-(2,4-diaminophenyl)diazen-1-yl]-3-[(1E)-2-[4-({4-[(1Z)-2-{7-[(1E)-2-(2,4-diaminophenyl)diazen-1-yl]-1-hydroxy-3-sulfonatonaphthalen-2-yl}diazen-1-yl]-2-sulfonatophenyl}amino)phenyl]diazen-1-yl]-4-hydroxynaphthalene-2-sulfonate
Details on test material:
Former EC 229-326-3

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium, other: TA 1538, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 1537
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
rat S9 and Hamster S9
Test concentrations with justification for top dose:
Replicate 01: 100, 333, 1000, 3333, 10000 µg/plate
Replicate 02: 1.0, 3.3, 10, 33, 100, 333, 1000, 3333, 10000 µg
PREINCUBATION METHODOLOGY UNDER REDUCTIVE CONDITIONS: 1.0, 3.3, 10, 33, 100, 333, 1000, 3333, 10000 µg
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: H2O
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Remarks:
PREINCUBATION METHODOLOGY UNDER REDUCTIVE CONDITIONS
Positive control substance:
congo red
Details on test system and experimental conditions:
CULTURE
Cultures were grown overnight in Oxoid nutrient broth no. 2 and were removed from incubation when they reached a density of (1-2) x 10^9 cells/mL. On the day of use, all tester strain cultures were checked for genetic integrity

S9 PREPARATION
Liver S9 homogenate was prepared from male Sprague-Dawley rats and Syrian golden hamsters that had been injected with Aroclor 1254 at 500 mg/kg body weight. The post-mitochondrial (microsomal) enzyme fractions were prepared.
The components of the S9 mix were:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP
100 mM sodium phosphate (pH 7.4)
and the appropriate S9 homogenate at a concentration of 0.1 mL/mL of mix.
For each plate receiving microsomal enzymes, 0.5 mL of S9 mix was added.

PLATE INCORPORATION METHODOLOGY.
For testing in the absence of S9 mix, 100 µL of the tester strain and 50 µL of the solvent or test chemical were added to 2.5 mL of molten selective top agar at 45 ± 2°C. When S9 was used, 0.5 mL of S9 mix, 50 µL of tester strain, and 50 µL of solvent or test chemical were added to 2.0 mL of molten selective top agar at 45 ± 2 °C.
After it was vortexed, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. After the overlay had solidified, the plates were incubated for 48 h at 37 ± 2°C. Five doses of test chemical, together with the appropriate concurrent solvent and positive controls, were tested in triplicate on each tester strain without metabolic activation and also with activation by induced rat and hamster liver S9 preparations.
The doses that were tested in the mutagenicity assay were selected based on the levels of cytotoxicity observed in a preliminary dose range-finding study using strain TA100. Ten dose levels of the chemical, one plate per dose, were tested in both the presence and the absence of induced hamster S9. If no toxicity was observed, a total maximum dose of 10 mg of test chemical per plate was used.

PREINCUBATION METHODOLOGY UNDER REDUCTIVE CONDITIONS
For tests using the FMN-modified assay, strains TA98 and TA100 were used. The bacteria, uninduced hamster liver S9 (30% v/v), cofactors (FMN, NADH, glucose-6-phosphate dehydrogenase, and glucose-6-phosphate), and test chemical were added, mixed, and incubated at 30 °C for 30 min without shaking. Nitrogen was blown over the preincubation tube to keep the atmosphere reduced. At the end of the incubation period, 2 mL of molten top agar was added to each sample tube and the mixture was poured on a minimal agar plate containing 0.5% glucose rather than the 2% glucose. The plates were then incubated at 37 °C for 48 h.
The positive control in all FMN experiments was Congo red. All plates were counted with an Artek automated colony counter (Artek 880, DynaTech, Chantilly, VA) or Minicount colony counter (Imaging Products International, Inc., Chantilly, VA), which was calibrated prior to use.
Evaluation criteria:
The criteria used to evaluate a test were as follows: for a test article to be considered positive, it had to induce at least a doubling (TA98, TA100, and TA1535) in the mean number of revertants per plate of at least one tester strain. This increase in the mean revertants per plate had to be accompanied by a dose response to increasing concentrations of the test chemical. If the study showed a dose response with a less than 3-fold increase on TA1537 or TA1538, the response had to be confirmed in a repeat experiment

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity

Any other information on results incl. tables

Dose TA98 TA100 TA1535 TA1537 TA1538
no S9 rat S9 Ham'r S9 no S9 rat S9 Ham'r S9 no S9 rat S9 Ham'r S9 no S9 rat S9 Ham'r S9 no S9 rat S9 Ham'r S9
REPLY 01   
Pos Pos Pos Neg Neg Pos Neg Neg Neg Neg Pos Pos Pos Pos Pos
H2O 20 ± 5 20 ± 3 27 ± 4 87 ± 6 88 ± 5 104 ± 10 17 ± 6 11 ± 2 6 ± 2 7 ± 3 5 ± 1 6 ± 3 12 ± 4 19 ± 3 18 ± 6
100ug 26 ± 2 97 ± 9 218 ± 22 94 ± 11 115 ± 3 102 ± 4 24 ± 1 10 ± 4 10 ± 3 7 ± 1 10 ± 4 18 ± 1 30 ± 5 172 ± 33 316 ± 15
333ug 38 ± 4 370 ± 18 814 ± 9 90 ± 19 167 ± 4 128 ± 9 20 ± 2 12 ± 2 16 ± 6 6 ± 1 26 ± 3 47 ± 6 46 ± 6 621 ± 34 996 ± 145
1000ug 62 ± 6 221 ± 60 346 ± 102 88 ± 10 101 ± 12 117 ± 19 20 ± 8 10 ± 2 7 ± 6 8 ± 2 21 ± 5 14 ± 2 87 ± 12 493 ± 77 807 ± 317
3333ug 73 ± 7 --- --- 94 ± 3 122 ± 20 120 ± 16 17 ± 6 9 ± 4 9 ± 2 9 ± 5 11 ± 4 13 ± 2 129 ± 19 --- ---
10000ug 79 ± 18 --- --- 93 ± 21 112 ± 18 122 ± 15 18 ± 3 8 ± 1 9 ± 3 12 ± 5 11 ± 3 13 ± 2 79 ± 16 --- ---
Positive 557 ± 21 718 ± 76 2136 ± 57 1191 ± 27 728 ± 76 2027 ± 113 1332 ± 188 386 ± 41 314 ± 8 492 ± 128 613 ± 19 238 ± 26 857 ± 124 808 ± 138 2233 ± 77
 REPLY 02
H2O 13 ± 4 15 ± 5 23 ± 2 101 ± 6 122 ± 14 156 ± 18 7 ± 1 7 ± 3 21 ± 4 28 ± 5
1.0ug --- 21 ± 5 39 ± 7 --- 123 ± 9 158 ± 6
3.3ug --- 40 ± 10 68 ± 7 --- 122 ± 2 174 ± 9 7 ± 3 6 ± 4 38 ± 7 42 ± 5
10ug 19 ± 2 46 ± 8 126 ± 10 105 ± 3 149 ± 12 165 ± 24 10 ± 1 10 ± 2 48 ± 9 61 ± 10
33ug 26 ± 2 153 ± 23 306 ± 99 99 ± 8 194 ± 10 195 ± 7 6 ± 2 8 ± 4 88 ± 12 118 ± 18
100ug 33 ± 2 238 ± 51 264 ± 67 94 ± 17 224 ± 21 258 ± 11 10 ± 3 15 ± 2 241 ± 33 331 ± 54
333ug 72 ± 4 320 ± 109 402 ± 121 119 ± 7 224 ± 29 350 ± 21 18 ± 2 46 ± 7 787 ± 39 1648 ± 75
1000ug 97 ± 11 115 ± 11 154 ± 47 121 ± 12 128 ± 20 189 ± 10 250 ± 4 373 ± 7 683 ± 142 1306 ± 204
3333ug 110 ± 10 96 ± 11 141 ± 14 127 ± 12 133 ± 11 164 ± 10
10000ug 93 ± 12 206 ± 68 173 ± 32 134 ± 10 131 ± 8 158 ± 6

PREINCUBATION METHODOLOGY UNDER REDUCTIVE CONDITIONS

Dose TA98 TA100
no S9 rat S9 Ham'r S9 no S9 rat S9 Ham'r S9
H2O 12 ± 1 25 ± 3 30 ± 5 --- --- 157 ± 8
1.0ug --- 29 ± 3 37 ± 8 --- --- 161 ± 11
3.3ug --- 38 ± 11 92 ± 1 --- --- 144 ± 12
10ug 21 ± 4 72 ± 11 188 ± 40 --- --- 183 ± 21
33ug 26 ± 5 221 ± 22 654 ± 105 --- --- 262 ± 13
100ug 59 ± 13 499 ± 45 1159 ± 101 --- --- 325 ± 8
333ug 86 ± 8 563 ± 107 1125 ± 47 --- --- 283 ± 25
1000ug 100 ± 15 135 ± 34 305 ± 113 --- --- 180 ± 11
3333ug 154 ± 4 117 ± 47 207 ± 91 --- --- 177 ± 5
10000ug 162 ± 14 115 ± 20 152 ± 25 --- --- 163 ± 18
Positive 391 ± 19 1410 ± 109 151 ± 159 --- --- 185 ± 23

Applicant's summary and conclusion

Conclusions:
Interpretation of results positive
Executive summary:

AMES test was performed on Direct Black 22 The experimental materials, methods, and procedures are based on those described by Ames et al. (Ames, B. N., McCann, J., and Yamasaki, E. (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test. Mutat. Res. 31, 347-364).

Result

P/3.3: positive