Chlorinated reaction product of Vat Blue 20 (Violanthrone A, Violanthrone B and Iso-violanthrone) - Earlier known as CI Vat Blue 18

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study Initiation : November 15, 2017 and Study Completion date : April 16, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Anthra[9,1,2-cde]benzo[rst]pentaphene-5,10-dione, chloro derivatives

EC Number:

215-378-4

EC Name:

Anthra[9,1,2-cde]benzo[rst]pentaphene-5,10-dione, chloro derivatives

Cas Number:

1324-54-5

Molecular formula:

C34H11Cl5O2

IUPAC Name:

Anthra[9,1,2-cde]benzo[rst]pentaphene-5,10-dione, chloro derivatives

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Constituent 3

Reference substance name:

Sodium chloride

EC Number:

231-598-3

EC Name:

Sodium chloride

Cas Number:

7647-14-5

Molecular formula:

ClNa

IUPAC Name:

sodium chloride

Test material form:

solid: particulate/powder

Details on test material:

Dark blue solid particulate/powder

Method

Target gene:

histidine

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 Fraction and S9 Mix

Test concentrations with justification for top dose:

5, 10, 20, 40, 80 and 160 µg/plate both in the absence and presence of metabolic activation system (10% v/v S9 mix)

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours of incubation at 37 ± 1°C
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays):

SPINDLE INHIBITOR (cytogenetic assays):

STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS:

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:

NUMBER OF CELLS EVALUATED:

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:

DETERMINATION OF CYTOTOXICITY
- cloning efficiency

- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):

- OTHER:

Evaluation criteria:

A result is considered positive if concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.
A response that did not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) was not evaluated as positive.
Negative results obtained in the initial toxicity-mutation test were confirmed by a second trial, using the same method as specified above, with an alteration in concentration spacing.

Statistics:

Simple linear regression analysis was performed for TA1537, TA1535, TA98, TA100 and TA102, separately, to assess the dose dependent nature of any increase in revertant colonies.

Results and discussion

Test results

Key result

Species / strain:

bacteria, other: TA1537, TA1535, TA98, TA100 and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: concluded to be non-mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium.

Applicant's summary and conclusion

Conclusions:

test item is concluded to be non-mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium.

Executive summary:

This study was performed by the bacterial reverse mutation test, using five histidine deficient mutant tester strains ofSalmonella typhimurium(i.e., TA1537, TA1535, TA98, TA100 and TA102).

Test substance was tested in two independent experiments, in the absence and presence of metabolic activation. Bacterial cultures were exposed totest itemat 8 concentrations (two plates/concentration) between 0.05 to 160 µg/plate in the initial toxicity-mutation test. Normal background lawn pattern was observed up to the tested concentration of 160 µg/plate in all tester strains (TA1537, TA1535, TA98, TA100 and TA102), no increase in the number of revertant colonies (no mutagenic effect) was observed both in the absence and presence of the metabolic activation system (5% v/v S9 mix) in all the tester strains. To confirm the negative results obtained in the initial toxicity mutation test, confirmatory mutation test was conducted with an increased S9 concentration i.e., 10% v/v S9 mix andmodified concentration spacing. Bacterial cultures were exposed totest itemat 6 concentrations (three plates/concentration) between 5 to 160 µg/plate both in the absence and presence (10% v/v S9 mix) of metabolic activation. After 48 hours of incubation at 37 ± 1°C, the revertant colonies were scored.

Test itemdid not induce any significant increase in the number of revertant colonies, in both trials, with and without S9 mix, in any of the five tester strains. All the values for the negative control were within historical control ranges of the laboratory and positive controls showed an increase in the number of revertant colonies, demonstrating the efficiency of the test system.

All criteria for a valid study were met as described in the study plan.From the results of this study, under the specified experimental conditions,test item is concluded to be non-mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium.