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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:
4.8.-21.9.2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was carried out in accordance with internationally valid GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
See Overall remarks
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: Fluidized Bed Combustion (FBC) Bottom Ash
- Substance type: technical product
- Physical state: solid
- Appearance: Light grey solid powder with some small black particle
- Chemical structure: Complex product of oxides
- Main components: SiO2 - 27,78%, Fe2O3 - 5,98%, CaO (total) - 35,65%, Na2O - 0,25%, P2O5 - 0,38%, CO2 - 0,5%, CaO (free) - 19,91%, Al2O3 - 11,16%, TiO2 - 2,65%, MgO - 0,44%, K2O - 0,42%, SO3 (sulphate) - 2,84%
- Impurities: Metals: As, Be, Cd, Co, Cr, Cu, Hg, Mo, Ni, Pb, Sb, Se, Tl, V, Zn - sum < 0,1%
- Lot/batch No.: FBC/230309/T2
- Expiration date of the lot/batch: 03/2024
- Storage: The substance will be stored in PE container at room temperature.
- Stability under test conditions: stable

Method

Target gene:
gene for synthesis histidine or tryptophan
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: histidine dependent strain
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: tryptophan dependent strain
Metabolic activation:
with and without
Metabolic activation system:
post-mitochondrial fraction (S9)
Test concentrations with justification for top dose:
50, 150, 500, 1000, 2500, 5000 µg per plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Water for injection, Ardeapharma a.s., Lot No. 0101030309
- Justification for choice of solvent/vehicle: solubility of the substance
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Migrated to IUCLID6: hydrochloride monohydrate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminofluorene
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: N-methyl-N´-nitro-N-nitrosoguanidine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)


NUMBER OF REPLICATIONS: Two series of experiments were performed with each strain - without metabolic activation and with a supernatant of rat liver and a mixture of cofactors.


DETERMINATION OF CYTOTOXICITY
- Method: total growth
Evaluation criteria:
The main criterion for evaluation of results was modified two-fold increase rule, its using is comparable with using of statistical methods. After this rule the result is positive, when reproducible dose-effect and/or doubling of ratio Rt/Rc is reached.
Statistics:
For the evaluation of results the modified two-fold increase rule was used, which using is comparable with application of statistical methods (2, 3).

2. Dunkel V. C., Chu K.C. (1980): Evaluation of methods for analysis of microbial mutagenicity assays, in The Predictive Value of Short-Term Screening Tests in Carcinogenicity Evaluation, Elsevier North-Holland Biomedical Press, 231 - 240
3. Claxton L. D. et al. (1987): Guide for the Salmonella typhimurium/mammalian microsome tests for bacterial mutagenicity, Mutat. Res. 189, 83 - 91

Results and discussion

Test resultsopen allclose all
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
Species / strain:
S. typhimurium TA 1537
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
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
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
without metabolic activation
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:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
First mutagenicity tests were performed with the same highest dose which was diluted according to the rules given in guidelines. No dificulties in evaluation showed at any dose, therefore the second mutagenicity experiments were done with the same doses.

COMPARISON WITH HISTORICAL CONTROL DATA:
Spontaneous reversions, negative controls (solvent), and positive controls were compared with historical controls in VUOS laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
As the test substance is almost insoluble in any solvent, a suspension was prepared in maximum concentration given in guidelines (5000 µg per plate). This concentration was further diluted and the concentration row arised (10-5000 µg per plate) was tested for toxicity in strain TA 100 without metabolic activation. No signs of toxicity were observed in any dose. Despite of the test substance particles on plates, evaluation was possible even at highest doses.
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 with metabolic activation
negative without metabolic activation

Under the above-described experimental design, the test substance Fluidized Bed Combustion (FBC) Bottom Ash was nonmutagenic for all the Salmonella typhimurium as well as Escherichia coli strains both in experiments without as well as with metabolic activation.
Executive summary:

Test substance Fluidized Bed Combustion (FBC) Bottom Ash was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The test was performed according to 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 and TA 1537 and one indicator Escherichia coli WP2 uvrA strain were used. The test substance was suspended in water for injection and assayed in doses of 50-5000 µg which were applied to plates in volume of 0.1 mL.

Two series of experiments were performed with each strain - without metabolic activation and with a supernatant of rat liver and a mixture of cofactors.

In the arrangement given above, the test substance Fluidized Bed Combustion (FBC) Bottom Ash was nonmutagenic for all the used bacterial strains with as well as without metabolic activation.