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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
Study period:
26 February to 06 March 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to OECD 471 and GLP guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2011

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
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Principles of method if other than guideline:
Due to the positive test result in the Initial Mutation Test (standard plate incorporation method), the same test method was used in the Confirmatory Mutation Test. The Prival modification was therefore skipped.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Details on test material:
Name: Reddish Blue

Method

Target gene:
Salmonella typhimurium:
TA98 hisD3052 Frameshift
TA100 hisG46 Base pair substitution
TA1535 hisG46 Base pair substitution
TA1537 hisC3076 Frameshift

Escherichia coli:
WP2uvrA trpE Base pair substitution
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
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: tryptophan dependent
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Test concentrations with justification for top dose:
Initial Mutation Test and Confirmatory Mutation Test : 250; 79.1; 25; 7.91; 2.5; 0.791; 0.25 µg/plate
Vehicle / solvent:
aqua bidest.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
w/o S9

Migrated to IUCLID6: TA100, TA1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
w/o S9

Migrated to IUCLID6: TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylene-diamine
Remarks:
TA98: w/o S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
w/o S9

Migrated to IUCLID6: WP2uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9: TA98, TA100, TA1535, TA1537, WP2uvrA

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
up to 500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
up to 500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
up to 500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: strain/cell type: plate incorporation test
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Spontaneous Reversion of Tester Strain Laboratory's historical control values for spontaneous revertants (revertants/plate) in the period of 1999 to 2007 (as guide) as follows: (-S9) Salmonella typhimuriumTA98: 10-54, TA100: 72-211, TA1535: 4-31, TA1537: 1-24, Escherichia coli WP2 uvrA: 9-66.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive Salmonella strains

In conclusion, the test item DYNS 2246 had strong mutagenic activity on the growth of the applied bacterium tester strains under the test conditions used in this study.
Executive summary:

The test item Reddish Blue was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains ofSalmonella typhimurium(Salmonella typhimuriumTA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain ofEscherichia coli(Escherichia coliWP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/b-naphthoflavone-induced rats (Preliminary Range Finding Test and Initial Mutation Test, Confirmatory Mutation Test) or from the livers of uninduced hamsters (Confirmatory Mutation Test).

The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Plate Incorporation Method or Pre-Incubation Method, Prival modification).

Based on the results of the Solubility Test, the test item was dissolved in DMF
(N,N-Dimethylformamide). Concentrations of5000; 2500; 1000; 316; 100; 31.6 and 10 µg/plate wereexamined in the Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in the independently performed main experiments (Initial Mutation Test and Confirmatory Mutation Test) were: 2500; 791; 250; 79.1; 25; 7.91 and 2.5 μg/plate.

Dose-related substantial increases were observed in both of the main tests inSalmonella typhimuriumTA98, TA100, TA1535 and TA1537 tester strains with and without metabolic activation. The observed effect was stronger inSalmonella typhimuriumTA98 and TA1537 bacterial strains, indicating that the mutations occurred mostly by frameshifts mutations.

Higher numbers of revertants compared to the solvent control were detected in theEscherichia coliWP2uvrAstrain without metabolic activation in the first main test; however, the calculated mutation factor values were below the biologically relevant threshold limit and the observed effect was not confirmed in the second main test.

Precipitate was detected on the plates at 2500 μg/plate concentration in all tester strains with and without metabolic activation in the Initial Mutation Test and Confirmatory Mutation Test.


The mean values of revertant colonies of the solvent control plates were within the historical control data range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. The tests were considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item induced gene mutations by base pair changes or frameshifts in the genome of the Salmonella strains used.

In conclusion, the test item Reddish Blue had mutagenic activity on the growth of the Salmonella typhimurium TA98, TA100, TA1535 and TA1537 tester strainsunder the test conditions used in this study. However, no mutagenic activity of the test item was confirmed on the Escherichia coli WP2uvrA bacterial strains under the test conditions of this study.