Custom Red #2

Genetic toxicity: in vitro

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:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): Custom Red #2
- Substance type: Violet powder
- Physical state: Solid
- Lot/batch No.: C50APR0805D
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark

Method

Target gene:

Histidine or Tryptophan gene

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

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): 1, 3, 10, 33, 100, 333, 1000 and 3330 µg/plate in the absence and presence of S9-mix
Experiment 1 (TA1535, TA1537, TA98): 3, 10, 33, 100 and 333 µg/plate in the absence and presence of S9-mix

Second mutation test:
TA1535, TA1537, TA98, TA100, WP2uvrA: 3, 10, 33, 100 and 333 µg/plate in the absence and presence of S9-mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test substance was not soluble in water, a workable suspension could be obtained in DMSO. Accepted and approved by authorities and international guidelines

Controls

Controls 1

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

Controls 2

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9

Migrated to IUCLID6: 5 µg/plate in saline for TA1535

Controls 3

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9

Migrated to IUCLID6: 10 µg/plate in DMSO for TA98

Controls 4

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9

Migrated to IUCLID6: 10 µg/plate in DMSO for WP2uvrA

Controls 5

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9

Migrated to IUCLID6: 60 µg/plate in water for TA1537

Controls 6

Positive controls:

yes

Positive control substance:

other: 2-amino anthracene: 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

NUMBER OF CELLS EVALUATED: 10E8 bacteria/plate

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

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).
-Reproducible increase in the mean number of revertant colonies is observed in both experiments.

Statistics:

No statistics

Results and discussion

Test results

Test results 1

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

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 2

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

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at dose levels of 333 µg/plate or above

RANGE-FINDING/SCREENING STUDIES: Yes


COMPARISON WITH HISTORICAL CONTROL DATA: Yes

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

It is concluded that this test is valid and that Custom Red #2 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.