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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06.09.2011 - 10.10.2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
In the first experiment in TA 1537, positive controls out of historical range occurred with as well as without metabolic activation. These experiments were repeated. This deviation had no impact on the outcome of the study.
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Ashes (residues), nonhazardous municipal solid waste
EC Number:
937-417-0
Molecular formula:
not applicable
IUPAC Name:
Ashes (residues), nonhazardous municipal solid waste
Test material form:
solid: bulk
Details on test material:
- Name of test material (as cited in study report): Ashes (residues), nonhazardous municipal solid waste
- Substance type: UVCB (inorganic oxides)
- Physical state: solid
- Composition of test material, percentage of components: SiO2 47.71 %; Al2O3 12.24 %; Fe2O3 9.62 %; CaO 15.80 %; Na2O 3.93 %; MgO 1.98 %; SO3 (total) 1.73 %; K2O 1.46 %; TiO2 1.22 %;
- Lot/batch No.: SPRUK-pololetí II/2010

Method

Target gene:
gene for synthesis histidine or tryptophan
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
other: aminoacid requiring
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of rat liver homogenate and mixture of cofactors
Test concentrations with justification for top dose:
50, 150, 500, 1500 and 5000 µg per plate
Vehicle / solvent:
Water for injection (Ardeapharma, lot No.0101181110)
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
AS
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminofluorene (CAS: 153-78-6)
Remarks:
2-AF
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (CAS: 613-13-8)
Remarks:
2-AA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 4-nitro-2-phenylenediamine (CAS: 99-56-9)
Remarks:
NPD
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
9-AAc
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine (CASRN: 70-25-7)
Remarks:
MNNG
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

TEST PROCEDURE: 100 µL of test substance of required concentration, 100 µL of 16-18 h culture of tester strain, 0.5 mL relevant buffer and 30 µl of S9 postmitochondrial 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. After incubation 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.

NUMBER OF REPLICATIONS: 3
Evaluation criteria:
The main criterion for evaluation of results was modified two-fold increase rule, which is compatible with the application of statistical methods (2, 3). 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“:
1. If the number of reversions is at least twice as high as that in the solvent control for the strains having spontaneous reversion >10;
2. 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.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Effect of the test substance

The Table is placed in Attached background material.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the experimental conditions, the test substance Ashes (residues), nonhazardous municipal solid waste was nonmutagenic for all the Salmonella typhimurium as well as Escherichia coli strains both in experiments without and with metabolic activation.
Executive summary:

Test substance Ashes (residues), nonhazardous municipal solid waste 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 coli WP2 uvrA strain were used. The test substance was suspended in water for injection and assayed in doses of (15) 50-5000µg, which were applied to plates in volume of 0.1 mL.

Experiments were performed without as well as with metabolic activation with a supernatant of rat liver and a mixture of cofactors.

In the arrangement given above, the test substance was nonmutagenic for all the used tester strains without as well as with metabolic activation.