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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item is considered to be not genotoxic in humans

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

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 / 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)

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.

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.

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.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 2016 - March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

The test item Vat Blue 6 was examined for ability to induce micronuclei in Chinese hamster V79 cells after in vitro treatment in the absence
and presence of S9 metabolism. The Chinese hamster V79 cell line is useful for this work because of its stable karyotype, short cell cycle and its high plating
efficiency. The most convenient stage to score micronuclei is the binucleate interphase stage. These
cells have completed one cell division after chemical treatment and are therefore capable
of expressing micronuclei. Treatment of cultures with the inhibitor of actin polymerisation
cytochalasin B blocks cytokinesis and cells that have completed one cell cycle after treatment
can be distinguished from non dividing cells by their binucleate appearance.

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:

Two independent assays for micronuclei induction were performed using dose levels:
Main Assay I (-/+S9): 60.0, 30.0, 15.0, 7.50, 3.75, 1.88, 0.938 and 0.469 μg/mL
Main Assay II (-S9): 30.0, 15.0, 7.50, 3.75, 1.88, 0.938, 0.469 and 0.234 μg/mL

Vehicle / solvent:

Test item solutions were prepared using dimethylsulfoxide (DMSO)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: Colchicine

Details on test system and experimental conditions:

The first main experiment was in the absence and presence of metabolic activation and involved short term treatment (3 hours).
The highest dose level to be used was determined according to the solubility of the test item in the vehicle and in the culture medium. Seven lower
dose levels, separated by a spacing of two were used. Approximately 20 hours before treatment, an appropriate number of flasks for the experiment
was prepared from a single pool of cells . Each 25 cm^2 flask was seeded with 500,000 cells in EMEM complete medium. The cells were allowed
to attach overnight prior to treatment in the incubator at 37°C in a 5% CO^2 atmosphere (100% nominal relative humidity).
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 twice with Phosphate Buffered Solution. Fresh culture medium and Cytochalasin B (3 µg/mL) were added and
the cultures were incubated for further 21 hours (Recovery Period) before harvesting. Cells were harvested at a time corresponding to
approximately 2 cell cycle lengths (25 hours).

In the second experiment, in the absence of S9 metabolic activation, flasks were seeded and prepared as described above. The treatment medium
was added to the flasks and after 3 hours of incubation at 37 °C, Cytochalasin B (3 µg/mL) was added to treatment media. The cultures were incubated at 37°C for further 21 hours (24-hours treatment).

Following treatment, the pH and osmolality of the treatment media were determined for
both experiments.
Appropriate negative and positive control cultures were included in the experiments. For
the short-term treatment (first experiment) the use of positive control was confined to
the clastogen Cyclophosphamide requiring metabolic activation. For the long-term treatment (second experiment), in the absence of S9
metabolism, positive control cultures were treated with the aneugen Colchicine.

At harvesting time the medium was removed from the flasks and the cells were brought into suspension by trypsinization. For each culture, the number of cells was adjusted to approximately 5x10^4 cells/mL. A suitable volume of each cell suspension was added to the cytofunnels in order to prepare slides using a CytospinTM. The cells were fixed in 90% methanol (-20°C). The slides were allowed to air dry and were kept at room temperature priorto staining. The slides were stained with a solution of Acridine Orange in PBS (12.5mg per 100mL PBS) and rinsed in PBS.
The cytokinesis-block proliferation index CBPI was calculated and was used to measure the cytotoxic effect. and the highest dose level for
genotoxicity assessment was selected on the basis of the cytotoxicity as calculated by the CBPI.
The highest dose level for genotoxicity assessment was selected as a dose which produces a substantial cytotoxicity compared with the solvent
control. Ideally the cytotoxicity should be between 40% and 50%. In the absence of cytotoxicity the highest treatment level was selected
as the highest dose level for scoring.
Two lower dose levels were also selected for the scoring of micronucleated cells.
For the three selected doses, for the solvent and positive controls (Colchicine and Cyclophosphamide),
2000 binucleated cells per cell culture were scored to assess the frequency of
micronucleated cells.
For the second main experiment, concerning cultures treated with Colchicine, since it is
a known mitotic spindle poison which induces mitotic slippage and cytokinesis block, a
greater magnitude of response was observed in mononucleated cells. For this reason 2000
mononucleated cells were scored.

The criteria for identifying micronuclei were as follows:
1. The micronucleus diameter was less than 1/3 of the nucleus diameter;
2. The micronucleus diameter was greater than 1/16 of the nucleus diameter;
3. No overlapping with the nucleus was observed;
4. The aspect was the same as the chromatin.

Evaluation criteria:

In this assay, the test item is considered as clearly positive if the following criteria are met:
– Significant increases in the proportion of micronucleated cells over the concurrent
controls occur at one or more concentrations.
– The proportion of micronucleated cells at such data points exceeds the normal range.
If the increases fall within the range of values normally observed in the negative control
cultures, the test item can not be classified as positive. Any significant increases over
the concurrent negative controls are therefore compared with historical control values
derived from recent studies.
– There is a significant dose effect relationship.

The test item is considered clearly negative if the following criteria are met:
– None of the dose level shows a statistically significant increase in the incidence of
micronucleated cells.
– There is no concentration related increase when evaluated with an appropriate trend
test.
– All the results are inside the distribution of the historical control data.

Statistics:

The numbers of binucleated cells with micronuclei in the control and treated cultures was
compared using a Chi-square method for the pooled cultures of each test point . The solvent
controls was used as the reference point for comparison in the statistical evaluation and the
evaluation of the results.
Cochran-Armitage Trend Test (one-sided) was performed to aid determination of concentration
response relationship.

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:

Cytotoxicity results:
The CBPI was calculated for each of the treatment series. Following treatment with the test item, no remarkable toxicity was observed in any
experiment, in the absence or presence of S9 metabolism. On the basis of these results, the dose levels selected for scoring of micronuclei were as
follows:
Main I (-/+S9): 60.0, 30.0, 15.0 µg/mL
Main II (-S9): 30.0, 15.0, 7.50 µg/mL

Assay results:
Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells was observed at
any dose level, in any treatment series and all the results were within the normal distribution range of historical control data. Slight increases in the
incidences of micronucleated cells over the concurrent control were observed in the second experiment. The incidences were within the normal distribution range of historical values for negative controls.
No concentration related increase of cells bearing micronuclei was observed in any experiment

Conclusions:

It is concluded that Vat Blue 6 does not induce micronuclei in Chinese hamster V79 cells after in vitro treatment, under the reported experimental
conditions.

Executive summary:

The test item Vat Blue 6 was assayed for the ability to induce micronuclei in Chinese hamster V79 cells, following in vitro treatment in the presence and absence of S9 metabolic activation. Two main experiments were performed. In the first experiment, the cells were treated for 3 hours in the absence and presence of S9 metabolism. The harvest time of 25 hours corresponding to approximately 2.0 cell cycles was used. During the solubility test, an opaque formulation with slight precipitation suitable for use was obtained with dimethylsulfoxide (DMSO) at the concentration of 6.00 mg/mL.On the basis of this result, a maximum dose level of 60.0 mg/mL was selected for the first main experiment by adding the test item suspension to treatment medium in the ratio 1:100. Lower dose levels corresponding to 30.0, 15.0, 7.50, 3.75, 1.88, 0.938 and 0.469 mg/mL were used. No cytotoxicity was observed at any dose level. As negative results were obtained, a second experiment was performed in the absence of S9 metabolism using a continuous treatment until harvest at 24 hours. For the second main experiment, using the long treatment in the absence of S9 metabolism, formulations of the test item were prepared at the same concentrations. However, due to the concurrent addition of the actin polymerisation inhibitor cytochalasin B to the treatment media (dissolved in DMSO), it was necessary to reduce the volume of test item solution in the treatment media to maintain DMSO concentration at 1%. Based on this statement and taking into account the results obtained in the first experiment, dose levels of 30.0, 15.0, 7.50, 3.75, 1.88, 0.938, 0.469 and 0.234 mg/mL were used for the second main experiment. Each experiment included appropriate negative and positive controls. Two cell cultures were prepared at each test point. Cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells. Dose levels were selected for the scoring of micronuclei on the basis of the cytotoxicity of the test item treatments calculated by the cytokinesis-block proliferation index (CBPI). Since no cytotoxicity was observed, the highest treatment level was selected as the highest dose level for scoring.

Dose levels selected for scoring were as follows:

Main I (-/+S9): 60.0, 30.0, 15.0 µg/mL

Main II (-S9): 30.0, 15.0, 7.50 µg/mL

One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells. Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the control value was observed at any dose level in any treatment series.

Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine, indicating the correct functioning of the test system. It is concluded that Vat Blue 6 does not induce micronuclei in Chinese hamster V79 cells after in vitro treatment, under the reported experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The test item was tested with the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 at concentrations from 2 to 300 µg per plate both in the presence and absence of metabolic activation. No mutagenic effect was observed

The test item Vat Blue 6 was assayed for the ability to induce micronuclei in Chinese hamster V79 cells, following in vitro treatment in the presence and absence of S9 metabolic activation. Two main experiments were performed. In the first experiment, the cells were treated for 3 hours in the absence and presence of S9 metabolism. The harvest time of 25 hours corresponding to approximately 2.0 cell cycles was used. During the solubility test, an opaque formulation with slight precipitation suitable for use was obtained with dimethylsulfoxide (DMSO) at the concentration of 6.00 mg/mL.On the basis of this result, a maximum dose level of 60.0 mg/mL was selected for the first main experiment by adding the test item suspension to treatment medium in the ratio 1:100. Lower dose levels corresponding to 30.0, 15.0, 7.50, 3.75, 1.88, 0.938 and 0.469 mg/mL were used. No cytotoxicity was observed at any dose level. As negative results were obtained, a second experiment was performed in the absence of S9 metabolism using a continuous treatment until harvest at 24 hours. For the second main experiment, using the long treatment in the absence of S9 metabolism, formulations of the test item were prepared at the same concentrations. However, due to the concurrent addition of the actin polymerisation inhibitor cytochalasin B to the treatment media (dissolved in DMSO), it was necessary to reduce the volume of test item solution in the treatment media to maintain DMSO concentration at 1%. Based on this statement and taking into account the results obtained in the first experiment, dose levels of 30.0, 15.0, 7.50, 3.75, 1.88, 0.938, 0.469 and 0.234 mg/mL were used for the second main experiment. Each experiment included appropriate negative and positive controls. Two cell cultures were prepared at each test point. Cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells. Dose levels were selected for the scoring of micronuclei on the basis of the cytotoxicity of the test item treatments calculated by the cytokinesis-block proliferation index (CBPI). Since no cytotoxicity was observed, the highest treatment level was selected as the highest dose level for scoring.

Dose levels selected for scoring were as follows:

Main I (-/+S9): 60.0, 30.0, 15.0 µg/mL

Main II (-S9): 30.0, 15.0, 7.50 µg/mL

One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells. Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the control value was observed at any dose level in any treatment series.

Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine, indicating the correct functioning of the test system. It is concluded that Vat Blue 6 does not induce micronuclei in Chinese hamster V79 cells after in vitro treatment, under the reported experimental conditions.

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.