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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.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

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:
03-FEB-2009 to 16-FEB-2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2010

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:
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
Details on test material:
Substance type: dark purple waxy solid
- Physical state: solid
- Stability under storage conditions: stable
- Storage condition of test material: at room temperature in the dark

Method

Target gene:
Histidine gene in S. typhimurium
Tryptophan gene in E. coli


TA100 hisG46/R-factor*
*: R-factor = plasmid pKM101 (increases error-prone DNA repair)
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
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
First mutation test
Dose range finding (TA100, WP2uvrA): A homogeneous suspension of the test substance could only be achieved at a maximum concentration of 20 mg/ml, therefore the following eight concentrations, 0.3, 1, 3, 10, 33, 100, 333 and 1000 µg/plate were tested in the absence and presence of S9-mix
Experiment 1 (TA1535, TA1537, TA98): 10, 33, 100, 333 and 1000 µg/plate in the absence of S9-mix
3, 10, 33, 100 and 333 µg/plate in the presence of S9-mix

Second mutation test:
TA1535, TA1537, TA98, TA100, WP2uvrA: 10, 33, 100, 333 and 1000 µg/plate in the absence of S9-mix
3, 10, 33, 100 and 333 µg/plate in the presence of S9-mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test substance was insoluble in water. No workable solution could be obtained in DMSO or ethanol. In acetone a workable suspension could be obtained at 20 mg/ml.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9

Migrated to IUCLID6: 650 µg/plate in DMSO for TA100
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9

Migrated to IUCLID6: 5 ↨0g/plate in saline for TA1535
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without S9

Migrated to IUCLID6: 10 µg/plate in DMSO for TA98
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9

Migrated to IUCLID6: 10 µg/plate in DMSO for WP2uvrA
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9

Migrated to IUCLID6: 60 µg/plate in water for TA1537
Positive control substance:
other: 2-amininoanthracene in DMSO for all strains
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION: incubation period: 48 Hours

NUMBER OF REPLICATIONS: 3 plates/treatment group

DETERMINATION OF CYTOTOXICITY:
- Method: The reduction of the bacterial background lawn and the reduction of the revertant colonies

OTHER EXAMINATIONS:
Precipitation of the test substance
Evaluation criteria:
The test material was considered positive if:
-A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
-The increase in the mean number of revertant colonies follows the concentration of test substance (dose-response relationship).

Statistics:
No statistics

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, but tested up to precipitating concentrations
Vehicle 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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At dose levels of 333 µg/plate and above

RANGE-FINDING/SCREENING STUDIES:
- No toxicity and mutagenicity was observed up to concentrations of 1000 µg/plate


COMPARISON WITH HISTORICAL CONTROL DATA: - The solvent and blank control and the strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Furthermore the results of the solvent control and the blank control were comparable for all strains in the absence and presence of S9-mix.
Remarks on result:
other: strain/cell type: Salmonella typhimurium
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that this test is valid and that Custom Red #3 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.