7,16-dichloro-6,15-dihydroanthrazine-5,9,14,18-tetrone

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January - May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Vat Blue 6

IUPAC Name:

Vat Blue 6

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Identity: Vat Blue 6
EC No.: 204-980-2
CAS no.: 130-20-1
Batch no.: 201405009
Content approximately: 100%
Empirical formula: C28H12Cl2N2O4
Molecular weight: 511.3 g/mol
Physical state: dark blue solid
Storage conditions: Room temperature protected from light
Expiry date: August 2019

Method

Target gene:

The test item Vat Blue 6 was examined for mutagenic activity by assaying for the induction of 6-
thioguanine resistant mutants in Chinese hamster V79 cells after in vitro treatment. 6-thioguanine can be
metabolised by the enzyme hypoxanthine-guaninphosphoribosyl-transferase (HPRT) into
nucleotides, which are used in nucleic acid synthesis resulting in the death of HPRT-competent cells. HPRTdeficient
cells, which are presumed to arise through mutations in the HPRT gene, cannot metabolise 6-thioguanine
and thus survive and grow in its presence.

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: EMEM medium supplemented with 10% Foetal Calf Serum (EMEM
complete)
- Properly maintained: yes; permanent stock of V79 cells are stored in liquid nitrogen and
subcoltures are prepared from the frozen stocks for experimental use.
- Periodically checked for Mycoplasma contamination: yes
- The karyotype, generation time, plating efficiency and mutation rates (spontaneous and induced)
have been checked in this laboratory.
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 tissue fraction: Species: Rat Strain: Sprague Dawley Tissue: Liver Inducing Agents: Phenobarbital – 5,6-Benzoflavone Producer: MOLTOX, Molecular Toxicology, Inc. Batch Number 3512

Test concentrations with justification for top dose:

A preliminary cytotoxicity assay was performed at the following dose levels: 60.0, 30.0, 15.0, 7.50, 3.75, 1.88, 0.938, 0.469 and
0.234 µg/mL.
Two independent assays for mutation to 6-thioguanine resistance were performed using dose levels:
Main Assay I (-/+S9): 60.0, 30.0, 15.0, 7.50 and 3.75 μg/mL
Main Assay II (-/+S9): 60.0, 37.5, 23.4, 14.6 and 9.13 μg/mL

Vehicle / solvent:

Test item solutions were prepared using dimethylsulfoxide (DMSO)

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

A preliminary cytotoxicity test was undertaken in order to select appropriate dose levels for the mutation assays. In
this test a wide range of dose levels of the test item was used. cell cultures were treated using the same treatment conditions as the mutation
assays, and the survival of the cells was subsequently determined. Treatments were performed both in the absence and presence of S9 metabolism; a
single culture was used at each test point and positive controls were not included.
Two Mutation Assays were performed including negative and positive controls, in the absence and presence of S9
metabolising system. Duplicate cultures were prepared at each test point, with the exception of the positive controls
which were prepared in a single culture. On the day before the experiment, sufficient numbers of 75 cm^2 flasks
were inoculated with 2 million freshly trypsinised V79 cells from a common pool. The cells were allowed to attach
overnight prior to treatment. Following treatment, the cultures were incubated at 37°C for three hours. At the end of
the incubation period, the treatment medium was removed and the cell monolayers were washed with PBS. Fresh
complete medium was added to the flasks which were then returned to the incubator at 37°C in a 5% CO^2
atmosphere (100% nominal relative humidity) to allow for expression of the mutant phenotype.

Determination of survival: The following day, the cultures were trypsinised and an aliquot was diluted and plated to
estimate the viability of the cells.

Subculturing: At Day 1 a number of cells was replated in order to maintain the treated cell populations. On Day 3,
the cell populations were subcultured in order to maintain them in exponential growth. When Day 9 is used as
expression time, subculturing was performed also at Day 6.

Determination of mutant frequency: A single expression time was used for each experiment: Day 6 in Main Assay I and Day 9 in
Main Assay II. At the expression time, each culture was trypsinised, resuspended in complete medium
and counted by microscopy. After dilution, an estimated 1 x 10^5 cells were plated in each of five 100 mm tissue
culture petri dishes containing medium supplemented with 6-thioguanine. These plates were subsequently stained
with Giemsa solutions and scored for the presence of mutants. After dilution, an estimated 200 cells were plated in
each of three 60 mm tissue culture petri dishes. These plates were used to estimate Plating Efficiency (P.E.).

Evaluation criteria:

For a test item to be considered mutagenic in this assay, it is required that:
- There is a five-fold (or more) increase in mutation frequency compared with the solvent controls, over two
consecutive doses of the test item. If only the highest practicable dose level (or the highest dose level not to cause
unacceptable toxicity) gives such an increase, then a single treatment-level will suffice.
- There must be evidence for a dose-relation (i.e. statistically significant effect in the ANOVA analysis).

Statistics:

The results of these experiments were subjected to an Analysis of Variance in which the effect of replicate culture
and dose level in explaining the observed variation was examined. For each experiment, the individual mutation
frequency values at each test point were transformed to induce homogeneous variance and normal distribution.
The appropriate transformation was estimated using the procedure of Snee and Irr (1981), and was found to be
y = (x + a)^b where a = 0 and b = 0.275. A two way analysis of variance was performed (without interaction) fitting to two
factors:
- Replicate culture: to identify differences between the replicate cultures treated.
- Dose level: to identify dose-related increases (or decreases) in response, after allowing for the effects of replicate
cultures and expression time.
The analysis was performed separately with the sets of data obtained in the absence and presence of S9
metabolism.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

Survival after treatment:
No relevant toxicity was observed at any concentration tested, in any experiment, in the
absence or presence of S9 metabolism.

Mutation results:
No relevant increases over the spontaneous mutation frequency were observed in any
experiment, at any treatment level in the absence or presence of S9 metabolic activation.
InMain Assay II, in the absence of S9 metabolic activation, a five-fold increase in mutation
frequency was observed at an intermediate dose level. However, mutation frequencies of
both replicates fell within the historical control range and the effect was not observed in
Main Assay I. Hence, the observed increase was considered an incidental event not related to
the action of the test item and of no biological significance. Analysis of variance indicated
that dose level and replicate culture were not significant factors in explaining the observed
variation in the data, in Main Assay I. In Main Assay II, dose level was a significant factor,
both in the absence and presence of S9 metabolism (p<0.05% and p<0.01%, respectively)
and replicate culture was a significant factor in its presence (p<0.05%). Mutation frequency
was in general higher in one of the replicate and at higher dose levels. However, all mutation
frequencies fell within the historical control range. Therefore the statistically significant
linear trend was considered to be attributable to an incidental event without any biological
relevance. In addition, heterogenity observed between replicate cultures has in no way
affected the validity of the experiment.

Applicant's summary and conclusion

Conclusions:

It is concluded that Vat Blue 6 does not induce mutation in Chinese hamster V79 cells after in vitro treatment, in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Executive summary:

The test item Vat Blue 6 was examined for mutagenic activity by assaying for the induction of 6-thioguanine resistant mutants in Chinese hamster V79 cells after in vitro treatment. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. Test item suspensions/solutions were prepared using dimethylsulfoxide (DMSO). A preliminary cytotoxicity assay was performed. Based on solubility features, the test item was assayed, in the absence and presence of S9 metabolism, at a maximum dose level of 60.0 µg/mL and at a wide range of lower dose levels: 30.0, 15.0, 7.50, 3.75, 1.88, 0.938, 0.469 and 0.234 µg/mL. By the end of treatment, precipitation of the test item was noted at the highest dose level, in the absence and presence of S9 metabolism. No toxicity was observed at any concentration tested, in the absence or presence of S9 metabolic activation. Two independent assays for mutation to 6-thioguanine resistance were performed using dose levels described in the following table:

Main Assay I (-/+S9): 60.0, 30.0, 15.0, 7.50 and 3.75 μg/mL

Main Assay II (-/+S9): 60.0, 37.5, 23.4, 14.6 and 9.13 μg/mL

The dose range used in Main Assay II was modified to focus on the highest concentrations that could be tested. No reproducible increases in mutant numbers or relevant five-fold increases in mutant frequency were observed following treatment with the test item at any dose level, in the absence or presence of S9 metabolism. Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. Marked increases in mutation frequency were obtained with the positive control treatments, indicating the correct functioning of the assay system. It is concluded that Vat Blue 6 does not induce gene mutation in Chinese hamster V79 cells after in vitro treatment, in the absence or presence of S9 metabolic activation, under the reported experimental conditions.