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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

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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
Study period:
1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study (OECD test guideline 471)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
2-methylundecan-2-aminium 1-amino-9,10-dioxo-4-[(2,4,6-trimethylphenyl)amino]-9,10-dihydroanthracene-2-sulfonate
EC Number:
700-958-6
Molecular formula:
UVCB Substance
IUPAC Name:
2-methylundecan-2-aminium 1-amino-9,10-dioxo-4-[(2,4,6-trimethylphenyl)amino]-9,10-dihydroanthracene-2-sulfonate
Details on test material:
- Description: blue powder
- Expiration date of the batch: 01-Apr-1997
-Stability under storage conditions: stable

Method

Target gene:
In the Salmonella typhimurium strains (TA 1535, TA 100, TA 1537, TA 98) the amino acid histidine locus is the target gene, in which induced back mutations will transform the histidine auxotrophy (his-) to histidine prototrophy (his+).
The Salmonella typhimurium histidine (his) reversion system measures his- => his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction from Aroclor 1254 induced male Wistar rats
Test concentrations with justification for top dose:
Experiment I: TA 98, TA 100, WP2 uvrA, and WP2 treated at 0, 3.3, 100.0, 333.3, 1000.0 and 5000.0 µg/plate
Experiment I: TA 1535 and TA 1537 treated at 0, 3.3, 100.0, 333.3, 666.6, 1000.0 and 5000.0 µg/plate

Experiment II: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA, and WP2 treated at 0, 3.3, 100.0, 333.3, 666.6, 1000.0 and 5000.0 µg/plate
Vehicle / solvent:
On the day of the experiment, the test item was dissolved in DMSO. The solvent was chosen because of its solubility properties and its relative non-toxicity for the bacteria.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
Solvent (DMSO)
True negative controls:
no
Positive controls:
yes
Remarks:
TA 1535, TA 100
Positive control substance:
sodium azide
Remarks:
without S9

Migrated to IUCLID6: 10 µg/plate
Positive controls:
yes
Remarks:
TA 1537, TA 98
Positive control substance:
other: 4-nitro-o-phenylene-diamine, 50 µg/plate
Remarks:
without S9
Positive controls:
yes
Remarks:
WP2 uvrA, WP2
Positive control substance:
methylmethanesulfonate
Remarks:
WP2 uvrA, WP2

Migrated to IUCLID6: 10 µL/plate
Positive controls:
yes
Remarks:
TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA, WP2
Positive control substance:
other: 2-aminoanthracene, 2.5 µg/plate
Details on test system and experimental conditions:
PRE-EXPERIMENT FOR TOXICITY
To evaluate the toxicity of the test item a pre-study was performed with strains TA 98, TA 100, WP2, and WP2 uvrA. 8 concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described below for the main experiment. Toxicity of the test item was evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures.

DOSE SELECTION
According to the results of the pre-experiment the concentrations applied in the main experiments were chosen. The concentration range covered two logarithmic decades. The maximum concentration was 5000.0 µg/plate. In experiment 1, 5 concentrations were documented from the pre-experiment with the strains TA 98, TA 100, WP2 uvrA, and WP2 and 6 concentrations were tested with the strains TA 1535 and TA 1537. In addition, 6 concentrations were tested in experiment 2 with all strains used. Two independent experiments were performed. As the results of the pre-experiment were in accordance with the criteria described above, these data were reported as a part of the main experiment 1.

EXPERIMENTAL PERFORMANCE
For each strain and dose level, including the controls, a minimum of three plates were used. The following materials were mixed in a test tube and poured onto the minimal agar plates:

100 µL test solution at each dose level, solvent control, negative control, or reference mutagen solution (positive control),
500 µL S9 mix (for test with metabolic activation) or S9 mix substitution-buffer (for test without metabolic activation),
100 µL bacteria suspension
2000 µL overlay agar

After solidification the plates were incubated upside-down for at least 48 hours at 37 ° C in the dark.

DATA RECORDING
The colonies were counted using the AUTOCOUNT (Artek Systems Corporation, BIOSYS GmbH, D-6367 Karben; F.R.G.). The counter was connected to an IBM AT compatible PC with printer which printed out the individual values and the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates. If precipitation of the test item precluded automatic counting the revertant colonies were counted by hand.
Evaluation criteria:
The generally accepted conditions for the evaluation of the results were:
- corresponding background growth on both negative control and test plates
- normal range of spontaneous reversion rates
A test item was considered as positive if either a dose related increase in the number of revertants or a significant and reproducible increase for at least one test concentration was induced. A test item producing neither a dose related increase in the number of revertants nor a significant and reproducible positive response at any one of the test points was considered non-mutagenic in this system.
A test item was considered as mutagenic if in the strains TA 100, WP2, and its uvrA derivate the number of reversions were at least twice as high and in the strains TA 1535, TA 1537, and TA 98 were at least three times higher as compared to the spontaneous reversion rate. Also, a dose-dependent increase in the number of revertants was regarded as an indication of possibly existing mutagenic potential of the test item regardless whether the highest dose induced the above described enhancement factors or not.
Statistics:
No appropriate statistical method is available.
Due to international guidelines a statistical evaluation of the results is recommended. However, no evaluated statistical procedure can be recommended for analysis of data from the bacterial assays at this time.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The background growth was reduced at 5000 µg/plate with and without S9 mix in experiment I and II in all strains used.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The background growth was reduced at 5000 µg/plate with and without S9 mix in experiment I and II in all strains used.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The background growth was reduced at 5000 µg/plate with and without S9 mix in experiment I and II in all strains used.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
A slight increase in revertant colony numbers was observed in strain E. coli WP2uvrA at 33.3 and 100.0 µg/plate without S9 mix in experiment II. This effect was considered not to be relevant since it could not be reproduced in the independent experiment.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative