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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study with acceptable restrictions (no data on test substance purity).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(adopted 21st July, 1997)
Deviations:
yes
Remarks:
(2-Aminoanthracene was used as the sole indicator of the efficacy of the S9-mix)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Disodium 4,4'-diamino-9,9',10,10'-tetrahydro-9,9',10,10'-tetraoxo[1,1'-bianthracene]-3,3'-disulphonate
EC Number:
227-877-4
EC Name:
Disodium 4,4'-diamino-9,9',10,10'-tetrahydro-9,9',10,10'-tetraoxo[1,1'-bianthracene]-3,3'-disulphonate
Cas Number:
6022-22-6
Molecular formula:
C28H16N2O10S2.2Na
IUPAC Name:
disodium 4,4'-diamino-9,9',10,10'-tetraoxo-9,9',10,10'-tetrahydro-1,1'-bianthracene-3,3'-disulfonate
Details on test material:
- Physical state: solid, red
- Analytical purity: no data
- Lot/batch No.: 00584CL6
- Expiration date of the lot/batch: April 01, 2011
- Storage condition of test material: room temperature

Method

Target gene:
his- and trp-gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat S9 liver microsomal fraction (induced with Phenobarbital and beta-Naphtoflavone)
Test concentrations with justification for top dose:
31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see: Details on test system and conditions
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation;

For the plate incorporation method (experiment I) the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
100 µL Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
100 µL Bacteria suspension,
2000 µL Overlay agar.

For the pre-incubation method (experiment II) 100 µL of the test item-preparation is preincubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 minutes at 37°C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.

After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.

The colonies were counted using a ProtoCOL counter (Meintrup DWS Laborgeraete GmbH). If precipitation of the test item precluded automatic counting the revertant colonies were counted by hand. In addition, tester strains with a low spontaneous mutation frequency like TA 1535 and TA 1537 were counted manually.

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately < 0.5 in relation to the solvent control

Positive Control Substances:
- Without metabolic activation:

- S. typhimurium: TA 100, TA 1535: Sodium azide, NaN3 (at least 99%) in aqua dest., 10 µg/plate;
- S. typhimurium: TA 98, TA 1537: 4-nitro-o-phenylene-diamine, 4-NOPD (> 97%) in DMSO, 10 µg/plate for TA98, 40 µg/plate for TA 1537
- E. coli: WP2 uvrA: Methyl methane sulfonate, MMS (99.0%) in Aqua dest., 1 µL/plate

- With metabolic activation:

- S. typhimurium: TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvrA: 2-aminoanthracene, 2-AA (97.5%) in DMSO, 2.5 µg/plate
Evaluation criteria:
Evaluation of Results:

The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to crystal violet and ampicillin
- the preculture shows a number of 10^9 cells/mL (quantified by optical density)
- the control plates without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range):

S. typhimurium:
- TA98: 18-63
- TA 100: 79-197
- TA 1535: 5-30
- TA1537: 4-32
E. coli:
- WP2uvrA: 38-122

- corresponding background growth on both negative control and test plates is observed.
- the positive controls show a distinct enhancement of revertant rates over the control plate.

A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 100 and WP2 uvrA the number of reversions is at least twice as high
- if in tester strains TA 1535, TA 1537 and TA 98 the number of reversions is at least three times higher as compared to the spontaneous reversion rate.

A test item 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.
Statistics:
According to the OECD guidelines, 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.

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:
no cytotoxicity
Remarks:
, but tested up to limit dose (= 5000 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
( 5000 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no

RANGE-FINDING/SCREENING STUDIES: The toxicity of the test item was determined with tester strains TA 98 and TA 100 in a pre-experiment. Eight concentrations were tested for toxicity and induction of mutations with 3 plates each. The experimental conditions in this pre-experiment were the same as described for the main experiment I (plate incorporation test). Toxicity may be detected by a clearing or rather diminution of the background
lawn or a reduction in the number of revertants down to a mutation factor of approximately < 0.5 in relation to the solvent control. The test item was tested in the pre-experiment at the following concentrations: 3.16, 10, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate. The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment. 5000 µg/plate was selected as the maximum concentration. Two independent experiments were performed at the following concentrations: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate. As the results of the pre-experiment were in accordance with the criteria described above, these were reported as a part of the main experiment I.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxic effects ofthe test item were noted in any of the five tester strains used up to the highest dose group evaluated (with and without metabolic activation) in experiment I and II.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

1st experiment: Standard plate test (31.6 - 5000 µg/plate)
Strain Metabolic activation system mean his+/trp+revertant colonies (negative control) maximum revertant factor (conc. (µg/plate)) dose dependency Assessment maximum revertant factor (positive control)
TA 98 no 24 1.0 (31.6/100/1000/2500/5000) no negative 18.1 (4-NOPD)
  yes 34 1.2 (31.6) no negative 56.4 (2-AA)
TA 100 no 120 1.0 (2500) no negative 18.7 (NaN3)
  yes 109 1.2 (2500) no negative 8.9 (2-AA)
TA 1537 no 15 1.3 (2500) no negative 13.6 (4-NOPD)
  yes 22 1.1 (2500) no negative 16.5 (2-AA)
TA 1535 no 19 0.9 (100/2500) no negative 49.1 (NaN3)
  yes 11 1.8 (31.6) no negative 12.9 (2-AA)
E. coli WP2 uvrA no 45 1.3 (1000) no negative 12.6 (MMS)
  yes 74 0.8 (316/1000/2500) no negative 2.8 (2-AA)
2nd experiment: Preincubation test (31.6 - 5000 µg/plate)
Strain Metabolic activation system mean his+/trp+revertant colonies (negative control) maximum revertant factor (conc. (µg/plate)) dose dependency Assessment maximum revertant factor (positive control)
TA 98 no 30 1.1 (31.6/316/5000) no negative 24.2 (4-NOPD)
  yes 54 1.1 (31.6) no negative 36.1 (2-AA)
TA 100 no 80 1.3 (31.6/100) no negative 4.7 (NaN3)
  yes 117 1.1 (316) no negative 16.1 (2-AA)
TA 1537 no 17 0.9 (316) no negative 10.9 (4-NOPD)
  yes 37 1.0 (31.6) no negative 7.4 (2-AA)
TA 1535 no 9 1.1 (31.6) no negative 54.0 (NaN3)
  yes 27 1.1 (31.6/1000) no negative 3.6 (2-AA)
E. coli WP2 uvrA no 51 1.0 (31.6/100/5000) no negative 12.6 (MMS)
  yes 67 1.1 (1000) no negative 2.6 (2-AA)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

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