N-[2-[(2-chloro-4,6-dinitrophenyl)azo]-5-(diallylamino)-4-methoxyphenyl]acetamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Only bacteria-specific effects were noted in the bacteria reverse mutation assay, As shown in the modified Ames Test with nitroreductase negative strains with a close structural analogue. This was confirmed by the mutagenicity study in mammalian cells with another structural analogue, which was negative.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read across is based on the same physico-chemical properties, a close structural similarity and the same mechanism of action during use processes.

2. SOURCE AND TARGET CHEMICAL(S)
Source: Structural Analogue 01
Target: Disperse Blue 291:1 Cl

3. ANALOGUE APPROACH JUSTIFICATION
see attachment section 13

4. DATA MATRIX
see attachment section 13

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Survival after treatment:
In Main Assay I, following treatment in the absence of S9 metabolism, a severe toxic effect was observed at the highest dose level (40.0 µg/mL) reducing survival to 2% of the concurrent negative control value. At the next lower concentration of 20.0 µg/mL, survival was reduced to 13%. Mild toxicity was observed at 10.0 µg/mL, while no relevant toxicity was noted at the remaining concentrations tested. In the presence of S9 metabolic activation, severe toxicity (relative survival < 10%) was observed at the two highest dose levels (600 and 400 µg/mL), while test item treatment at 267 µg/mL yielded 41% relative survival. At lower dose levels, no relevant toxicity was noted. At the highest dose level tested, no cells were recovered on Day 6 both in the absence and presence of S9 metabolic activation. Since at low survival levels, mutation results are prone to a number of artefacts (selection effects, sampling error, founder effects) and mechanisms other than direct genotoxicity per se can lead to positive results (e.g. events associated with apoptosis, endonuclease release from lysosomes, etc.), the dose level of 400 µg/mL was excluded from analysis.
In Main Assay II, in the absence of S9 metabolism, survival was reduced to 15% at the highest dose level of 20.0 µg/mL; moderate toxicity (approximately 30% of RS) was noted at the two next lower concentrations; mild toxicity (RS=58%) was observed at 9.10 µg/mL, while no relevant toxicity was observed at the remaining dose levels. It should be noted that on Day 6 a lower number of cells, compared to the negative control value, was recovered at the highest dose level. In the presence of S9 metabolism, test item treatment at 400 µg/mL yielded 31% relative survival; mild toxicity (RS = 63%) was noted at the next lower dose level, while no relevant toxicity was observed over the remaining concentrations.
Mutation results:
In the presence of S9 metabolism, no increase over the spontaneous mutation frequency was observed in any experiment, at any treatment level. In the absence of S9 metabolic activation, no increase over the spontaneous mutation frequency was observed in Main assay I (Day 8), while a dose related increase in mutation frequency, which reached five-fold the concurrent negative control value at 15.4 µg/mL, was observed in Main Assay II (Day 6). Analysis of variance indicated that replicate culture was not a significant factor in explaining the observed variation in the data, in the absence and presence of S9 metabolism, in Main Assay I and II. Dose level was a significant factor (p < 0.05%) in explaining the observed variation in the data of Main Assay II in the absence of S9 metabolism.

Conclusions:

The substance 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 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 solutions were prepared using dimethylsulfoxide (DMSO). A preliminary cytotoxicity assay was performed, in the absence and presence of S9 metabolic activation. Based on solubility features, the test item was assayed at a maximum dose level of 1250 µg/mL and at a wide range of lower dose levels: 625, 313, 156, 78.1, 39.1, 19.5, 9.77 and 4.88 µg/mL. In the absence of S9 metabolism, no cells survived at almost all the concentrations tested; survival was reduced to 50% of the concurrent negative control value at the lowest dose level (4.88 µg/mL). In the presence of S9 metabolism, severe toxicity was noted at the two highest concentrations, while survival was reduced to 44% of the concurrent negative control value at 313 µg/mL. No relevant toxicity was noted over the remaining concentrations tested. By the end of treatment time, precipitation of test item and/or a coloured film, adhering to the flask surface, was observed at all concentrations tested in the absence of S9 metabolism and at the three highest dose levels in its presence. In order to select the appropriate range of concentrations for the mutation assay, an additional toxicity test was performed in the absence of S9 metabolism using the following lower and closer concentrations: 10.0, 7.14, 5.10, 3.64 and 2.60 µg/mL. Since no adequate toxicity for dose selection was obtained, a third toxicity test was performed in the absence of S9 metabolism, using the following concentrations: 40.0, 20.0, 10.0, 5.00 µg/mL. Severe toxicity was noted at the highest concentration; survival was reduced to 12% and 37% of the concurrent negative control value at 20.0 and 10.0 µg/mL, respectively; while no relevant toxicity was observed at 5.00 µg/mL. Two independent assays for mutation to 6-thioguanine resistance were performed using the following dose levels:

Main Assay I (+S9): 600, 400, 267, 178, 119 and 79.0 µg/mL

Main Assay I (-S9): 40.0, 20.0, 10.0, 5.00, 2.50, 1.25 and 0.625 µg/mL

Main Assay II (+S9): 400, 308, 237, 182, 140 and 108 µg/mL

Main Assay II (+S9): 20.0, 15.4, 11.8, 9.10, 7.00 and 5.39 µg/mL

In the second experiment the concentration range was slightly modified on the basis of the toxicity results obtained in Main Assay I. No reproducible five-fold increases in mutant numbers or 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 were obtained with the positive control treatments indicating the correct functioning of the assay system.

It is concluded that the substance 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 gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 December 2014 to 04 May 2015

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 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. HPRT-deficient 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 Numbers. 3332, 3263 and 3417

Test concentrations with justification for top dose:

A preliminary cytotoxicity assay was performed at the following dose levels: 1250, 625, 313, 156, 78.1, 39.1, 19.5, 9.77 and 4.88 µg/mL.
A second toxicity test was performed in the absence of S9 metabolism using the following lower and closer concentrations: 10.0, 7.14, 5.10, 3.64 and 2.60 µg/mL.
Since no adequate toxicity for dose selection was obtained, a third toxicity test was performed in the absence of S9 metabolism, using the following concentrations: 40.0, 20.0, 10.0, 5.00 µg/mL.
Two independent assays for mutation to 6-thioguanine resistance were performed using dose levels:
Main Assay I (+S9): 600, 400, 267, 178, 119 and 79.0 µg/mL
Main Assay I (-S9): 40.0, 20.0, 10.0, 5.00, 2.50, 1.25 and 0.625 µg/mL
Main Assay II (+S9): 400, 308, 237, 182, 140 and 108 µg/mL
Main Assay II (+S9): 20.0, 15.4, 11.8, 9.10, 7.00 and 5.39 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:

Preliminary cytotoxicity tests were undertaken in order to select appropriate dose levels for the mutation assays: a single experiment was performed in the presence of S9 metabolism, while three tests were performed in the absence of S9 metabolic activation.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 cm2 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% CO2 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: On Day 3, the cell populations were subcultured in order to maintain them in exponential growth. When Day 8 was used as expression time, subculturing was performed on Day 4 and Day 6.
Determination of mutant frequency: A single expression time was used for each experiment: Day 8 in Main Assay I and Day 6 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:

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.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Survival after treatment:
In Main Assay I, following treatment in the absence of S9 metabolism, a severe toxic effect was observed at the highest dose level (40.0 µg/mL) reducing survival to 2% of the concurrent negative control value. At the next lower concentration of 20.0 µg/mL, survival was reduced to 13%. Mild toxicity was observed at 10.0 µg/mL, while no relevant toxicity was noted at the remaining concentrations tested. In the presence of S9 metabolic activation, severe toxicity (relative survival < 10%) was observed at the two highest dose levels (600 and 400 µg/mL), while test item treatment at 267 µg/mL yielded 41% relative survival. At lower dose levels, no relevant toxicity was noted. At the highest dose level tested, no cells were recovered on Day 6 both in the absence and presence of S9 metabolic activation. Since at low survival levels, mutation results are prone to a number of artefacts (selection effects, sampling error, founder effects) and mechanisms other than direct genotoxicity per se can lead to positive results (e.g. events associated with apoptosis, endonuclease release from lysosomes, etc.), the dose level of 400 µg/mL was excluded from analysis.
In Main Assay II, in the absence of S9 metabolism, survival was reduced to 15% at the highest dose level of 20.0 µg/mL; moderate toxicity (approximately 30% of RS) was noted at the two next lower concentrations; mild toxicity (RS=58%) was observed at 9.10 µg/mL, while no relevant toxicity was observed at the remaining dose levels. It should be noted that on Day 6 a lower number of cells, compared to the negative control value, was recovered at the highest dose level. In the presence of S9 metabolism, test item treatment at 400 µg/mL yielded 31% relative survival; mild toxicity (RS = 63%) was noted at the next lower dose level, while no relevant toxicity was observed over the remaining concentrations.
Mutation results:
In the presence of S9 metabolism, no increase over the spontaneous mutation frequency was observed in any experiment, at any treatment level. In the absence of S9 metabolic activation, no increase over the spontaneous mutation frequency was observed in Main assay I (Day 8), while a dose related increase in mutation frequency, which reached five-fold the concurrent negative control value at 15.4 µg/mL, was observed in Main Assay II (Day 6). Analysis of variance indicated that replicate culture was not a significant factor in explaining the observed variation in the data, in the absence and presence of S9 metabolism, in Main Assay I and II. Dose level was a significant factor (p < 0.05%) in explaining the observed variation in the data of Main Assay II in the absence of S9 metabolism.

Conclusions:

The test substance 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 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 solutions were prepared using dimethylsulfoxide (DMSO). A preliminary cytotoxicity assay was performed, in the absence and presence of S9 metabolic activation. Based on solubility features, the test item was assayed at a maximum dose level of 1250 µg/mL and at a wide range of lower dose levels: 625, 313, 156, 78.1, 39.1, 19.5, 9.77 and 4.88 µg/mL. In the absence of S9 metabolism, no cells survived at almost all the concentrations tested; survival was reduced to 50% of the concurrent negative control value at the lowest dose level (4.88 µg/mL). In the presence of S9 metabolism, severe toxicity was noted at the two highest concentrations, while survival was reduced to 44% of the concurrent negative control value at 313 µg/mL. No relevant toxicity was noted over the remaining concentrations tested. By the end of treatment time, precipitation of test item and/or a coloured film, adhering to the flask surface, was observed at all concentrations tested in the absence of S9 metabolism and at the three highest dose levels in its presence. In order to select the appropriate range of concentrations for the mutation assay, an additional toxicity test was performed in the absence of S9 metabolism using the following lower and closer concentrations: 10.0, 7.14, 5.10, 3.64 and 2.60 µg/mL. Since no adequate toxicity for dose selection was obtained, a third toxicity test was performed in the absence of S9 metabolism, using the following concentrations: 40.0, 20.0, 10.0, 5.00 µg/mL. Severe toxicity was noted at the highest concentration; survival was reduced to 12% and 37% of the concurrent negative control value at 20.0 and 10.0 µg/mL, respectively; while no relevant toxicity was observed at 5.00 µg/mL. Two independent assays for mutation to 6-thioguanine resistance were performed using the following dose levels:

Main Assay I (+S9): 600, 400, 267, 178, 119 and 79.0 µg/mL

Main Assay I (-S9): 40.0, 20.0, 10.0, 5.00, 2.50, 1.25 and 0.625 µg/mL

Main Assay II (+S9): 400, 308, 237, 182, 140 and 108 µg/mL

Main Assay II (+S9): 20.0, 15.4, 11.8, 9.10, 7.00 and 5.39 µg/mL

In the second experiment the concentration range was slightly modified on the basis of the toxicity results obtained in Main Assay I. No reproducible five-fold increases in mutant numbers or 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 were obtained with the positive control treatments indicating the correct functioning of the assay system.

It is concluded that the substance 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 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Plate incorporation test under oxidative conditions according to Maron and Ames (1983), with modifications, using sterile deionized water as the negative control.
All the experiments were performed in duplicate. The dye was tested under partially anaerobic (reducing) conditions, and the results were compared to the data obtained using the normal oxidative protocol. The assay under reductive conditions was performed according to Prival and Mitchell (1982).
The mutation spectra of CI Disperse Blue 291 were determined using the Salmonella TA7000 series described by Gee et al. (1994) using the microsuspension modification (Kado et al., 1983) under oxidative conditions. Overnight cultures of each strain (around 109 cells/ml) were concentrated 10 fold by centrifugation at 10,000g at 4 °C for 10 min and resuspended in 0.015 M sodium phosphate buffer. 50 µI of cell suspension, 50 µI of 0.015 M sodium phosphate buffer or S9 mix, and 7.5 µI of the test sample were added to a tube and incubated at 37 °C for 90 min without shaking. After incubation, 2 ml of molten agar was added, the mixture was poured onto a minimal agar plate, and the plates were incubated at 37 °C for 72 h. Colonies were counted using an automatic colony counter. When the number of colonies was less than ten, the counts were also confirmed by hand, using a light box with magnification.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Genotype and the type of mutation detected by each strain of Salmonella typhimurium

Strains of Salmonella. Genotype Type of mutation detected Reference
TA1535 HisG46, rfa, Abio, AuvrB Frameshift (mainly CG deletions) Maron and Ames (1983)
TA1537 HisC3076, rfa, Abio, AuvrB Frameshift (mainly CG deletions) Maron and Ames (1983)
TA1538 HisD3052, rfa, Abio, AuvrB Frameshift (mainly CG deletions) Maron and Ames (1983)
TA98 HisD3052, rfa, Abio, AuvrB, pKM101 Frameshift (mainly CG deletions) Maron and Ames (1983)
TA98 NR HisD3052, rfa, Abio, AuvrB, pKM101 nitroreductase enzyme activity deficient Frameshift (mainly CG deletions) Rosenkranz and Mermelstein (1983)
TA98 DNP6 HisD3052, rfa, Abio, AuvrB, pKM101 acetyltransferase enzyme activity deficient Frameshift (mainly CG deletions) Rosenkranz and Mermelstein (1983)
TA100 HisG46, rfa, Abio, AuvrB, pKM101 Missence (base pair substitutions) Maron and Ames (1983)
YG1021 HisD3052, rfa, Abio, AuvrB, pKM101, nitroreductase overproducing enzyme activity Frameshift (mainly CG deletions) Watanabe et al. (1989)
YG1024 HisD3052, rfa, Abio, AuvrB, pKM101, acetyltransferase overproducing enzyme activity Frameshift (mainly CG deletions) Watanabe et al. (1990)
YG1041 HisD3052, rfa, Abio, AuvrB, pKM101, acetyltransferase and nitroreductase overproducing enzyme activity Frameshift (mainly CG deletions) Hagiwara et al. (1993)
TA7001 IlisG1775, rfa, Abio, AuvrB, pKM101 T:A —> C:G Gee et al. (1994)
TA7002 IlisC9138, rfa, Abio, AuvrB, pKM101 T:A —> A:T Gee et al. (1994)
TA7003 HisG9074, rfa, Abio, AuvrB, pKM101 T:A —> G:C Gee et al. (1994)
TA7004 IlisG9133, rfa, Abio, AuvrB, pKM101 C:G —> T:A Gee et al. (1994)
TA7005 IlisG9130, rfa, Abio, AuvrB, pKM101 C:G —> A:T Gee et al. (1994)
TA7006 HisG9070, rfa, Abio, AuvrB, pKM101 C:G —> G:C Gee et al. (1994)

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium, other: TA98 NR

Additional strain / cell type characteristics:

nitroreductase deficient

Species / strain / cell type:

S. typhimurium, other: TA98 DNP6

Additional strain / cell type characteristics:

acetyltransferase deficient

Species / strain / cell type:

S. typhimurium, other: YG1021

Additional strain / cell type characteristics:

other: nitroreductase overproducing enzyme activity

Species / strain / cell type:

S. typhimurium, other: YG1024

Additional strain / cell type characteristics:

acetyltransferase proficient

Species / strain / cell type:

S. typhimurium, other: YG1041

Additional strain / cell type characteristics:

other: acetyltransferase and nitroreductase overproducing enzyme activity

Species / strain / cell type:

S. typhimurium, other: TA7001, TA7002, TA7003, TA7004, TA7005, TA7006

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain / cell type:

S. typhimurium TA 98

Metabolic activation:

with and without

Metabolic activation system:

Sprague Dawley rat liver S9 (MolTox, Boone, NC) induced with an Arochlor 1254 mix

Test concentrations with justification for top dose:

up to 500 µg/plate
TA 7000 stains: 250, 500, 1000 µg/plate

Vehicle / solvent:

sterile deionized water and DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Evaluation criteria:

Samples were considered positive when a significant ANOVA and dose response was obtained with the Bernstein model (Bernstein et al., 1982). For the TA7000 strain series, because of the low spontaneous mutation rate of some strains, we considered a response positive when the counts of the plates containing the sample were greater than the average of the negative controls plus 3 standard deviations. The results were expressed as number of reverants per µg of compound tested.

Statistics:

ANOVA

Species / strain:

S. typhimurium, other: TA1537, TA100, TA98, TA1538

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA98NR

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA98NR

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: YG1021, YG1024, YG1041

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA98DNP6

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA98DNP6

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

slight increase

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA7001, TA7002, TA7004, TA7005

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

TA7004, TA7005 at 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA7003, TA7006

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

TA7006 at 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA7000 series

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

under reductive cleavage conditions, the color of the tested dye did not change when using the modification of Prival and Mitchell (1982), and the mutagenicity remained the same as that seen with normal oxidative metabolism. This suggests that the azobond was not cleaved in the testing conditions.

Conclusions:

The study shows that the mutagenic effect of the test substance is due to bacterial nitroreductase and O-acetyltransferase activity

Executive summary:

Disperse Blue 291 was tested for mutagenic activity in the Salmonella assay with strains with different levels of nitroreductase and O-acetyltransferase (i.e., TA98DNP6, YG1024, and YG1041) as well as standard strains TA 1535, TA1537, TA1538, TA98 and TA100 and strains which provide more information on the base-pair substitution (TA 7001 to 7006). Disperse Blue 291 showed direct-acting mutagenic activity with all strains ofSalmonella typhimuriumtested, except forTA 1535.According to the classification of Claxton et al. (1991), the potency of this product can be considered moderate (10-100 revertants/µg). In the absence of S9, the nitroreduction is strongly related to the mutagenic activity, because the mutagenicity of Disperse Blue 291 was very low when tested with the strains lacking nitroreductase activity (TA98NR) and was increased with the nitroreductase overproducing strains, (YG1021 and YG1041). The same mutagenic pattern was observed for the acetyltransferase deficient and overproducing strains (TA98DNP6, YG1024, and YG1041) revealing also the importance of the acetyltransferase enzyme in the activation of Disperse Blue 291. Because of the remarkable increase in the response with the nitroreductase andO-acetyltransferase overproducing strain (YG1041), it is likely that the product of the nitroreductase is a substrate for theO-acetyltransferase.

In the presence of S9, the mutagenicity was slightly increased with TA98NR, TA98, YG1021, TA98DNP6, and YG1024 suggesting that, P450 enzymes also have a role in the activation of these compounds, besides the bacterial enzymes. This could be explained by the activation of other radicals of the molecule by the S9 enzymes, for example the —OCH₃or the —N(CH₂CH₃)₂of the CI Disperse Blue 291.

 

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read across is based on the same physico-chemical properties, a close structural similarity and the same mechanism of action during use processes.

2. SOURCE AND TARGET CHEMICAL(S)
Source: Disperse Blue 291 Br
Target: Disperse Blue 291:1 Cl

3. ANALOGUE APPROACH JUSTIFICATION
see attachment section 13

4. DATA MATRIX
see attachment section 13

Reason / purpose for cross-reference:

read-across source

Principles of method if other than guideline:

Plate incorporation test under oxidative conditions according to Maron and Ames (1983), with modifications, using sterile deionized water as the negative control.
All the experiments were performed in duplicate. The dye was tested under partially anaerobic (reducing) conditions, and the results were compared to the data obtained using the normal oxidative protocol. The assay under reductive conditions was performed according to Prival and Mitchell (1982).
The mutation spectra of CI Disperse Blue 291 were determined using the Salmonella TA7000 series described by Gee et al. (1994) using the microsuspension modification (Kado et al., 1983) under oxidative conditions. Overnight cultures of each strain (around 109 cells/ml) were concentrated 10 fold by centrifugation at 10,000g at 4 °C for 10 min and resuspended in 0.015 M sodium phosphate buffer. 50 µI of cell suspension, 50 µI of 0.015 M sodium phosphate buffer or S9 mix, and 7.5 µI of the test sample were added to a tube and incubated at 37 °C for 90 min without shaking. After incubation, 2 ml of molten agar was added, the mixture was poured onto a minimal agar plate, and the plates were incubated at 37 °C for 72 h. Colonies were counted using an automatic colony counter. When the number of colonies was less than ten, the counts were also confirmed by hand, using a light box with magnification.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Genotype and the type of mutation detected by each strain of Salmonella typhimurium

Strains of Salmonella. Genotype Type of mutation detected Reference
TA1535 HisG46, rfa, Abio, AuvrB Frameshift (mainly CG deletions) Maron and Ames (1983)
TA1537 HisC3076, rfa, Abio, AuvrB Frameshift (mainly CG deletions) Maron and Ames (1983)
TA1538 HisD3052, rfa, Abio, AuvrB Frameshift (mainly CG deletions) Maron and Ames (1983)
TA98 HisD3052, rfa, Abio, AuvrB, pKM101 Frameshift (mainly CG deletions) Maron and Ames (1983)
TA98 NR HisD3052, rfa, Abio, AuvrB, pKM101 nitroreductase enzyme activity deficient Frameshift (mainly CG deletions) Rosenkranz and Mermelstein (1983)
TA98 DNP6 HisD3052, rfa, Abio, AuvrB, pKM101 acetyltransferase enzyme activity deficient Frameshift (mainly CG deletions) Rosenkranz and Mermelstein (1983)
TA100 HisG46, rfa, Abio, AuvrB, pKM101 Missence (base pair substitutions) Maron and Ames (1983)
YG1021 HisD3052, rfa, Abio, AuvrB, pKM101, nitroreductase overproducing enzyme activity Frameshift (mainly CG deletions) Watanabe et al. (1989)
YG1024 HisD3052, rfa, Abio, AuvrB, pKM101, acetyltransferase overproducing enzyme activity Frameshift (mainly CG deletions) Watanabe et al. (1990)
YG1041 HisD3052, rfa, Abio, AuvrB, pKM101, acetyltransferase and nitroreductase overproducing enzyme activity Frameshift (mainly CG deletions) Hagiwara et al. (1993)
TA7001 IlisG1775, rfa, Abio, AuvrB, pKM101 T:A —> C:G Gee et al. (1994)
TA7002 IlisC9138, rfa, Abio, AuvrB, pKM101 T:A —> A:T Gee et al. (1994)
TA7003 HisG9074, rfa, Abio, AuvrB, pKM101 T:A —> G:C Gee et al. (1994)
TA7004 IlisG9133, rfa, Abio, AuvrB, pKM101 C:G —> T:A Gee et al. (1994)
TA7005 IlisG9130, rfa, Abio, AuvrB, pKM101 C:G —> A:T Gee et al. (1994)
TA7006 HisG9070, rfa, Abio, AuvrB, pKM101 C:G —> G:C Gee et al. (1994)

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium, other: TA98 NR

Additional strain / cell type characteristics:

nitroreductase deficient

Species / strain / cell type:

S. typhimurium, other: TA98 DNP6

Additional strain / cell type characteristics:

acetyltransferase deficient

Species / strain / cell type:

S. typhimurium, other: YG1021

Additional strain / cell type characteristics:

other: nitroreductase overproducing enzyme activity

Species / strain / cell type:

S. typhimurium, other: YG1024

Additional strain / cell type characteristics:

acetyltransferase proficient

Species / strain / cell type:

S. typhimurium, other: YG1041

Additional strain / cell type characteristics:

other: acetyltransferase and nitroreductase overproducing enzyme activity

Species / strain / cell type:

S. typhimurium, other: TA7001, TA7002, TA7003, TA7004, TA7005, TA7006

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain / cell type:

S. typhimurium TA 98

Metabolic activation:

with and without

Metabolic activation system:

Sprague Dawley rat liver S9 (MolTox, Boone, NC) induced with an Arochlor 1254 mix

Test concentrations with justification for top dose:

up to 500 µg/plate
TA 7000 stains: 250, 500, 1000 µg/plate

Vehicle / solvent:

sterile deionized water and DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Evaluation criteria:

Samples were considered positive when a significant ANOVA and dose response was obtained with the Bernstein model (Bernstein et al., 1982). For the TA7000 strain series, because of the low spontaneous mutation rate of some strains, we considered a response positive when the counts of the plates containing the sample were greater than the average of the negative controls plus 3 standard deviations. The results were expressed as number of reverants per µg of compound tested.

Statistics:

ANOVA

Species / strain:

S. typhimurium, other: TA1537, TA100, TA98, TA1538

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA98NR

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA98NR

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: YG1021, YG1024, YG1041

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA98DNP6

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA98DNP6

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

slight increase

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA7001, TA7002, TA7004, TA7005

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

TA7004, TA7005 at 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA7003, TA7006

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

TA7006 at 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA7000 series

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

under reductive cleavage conditions, the color of the tested dye did not change when using the modification of Prival and Mitchell (1982), and the mutagenicity remained the same as that seen with normal oxidative metabolism. This suggests that the azobond was not cleaved in the testing conditions.

Conclusions:

The study shows that the mutagenic effect of the test substance is due to bacterial nitroreductase and O-acetyltransferase activity

Executive summary:

Disperse Blue 291 was tested for mutagenic activity in the Salmonella assay with strains with different levels of nitroreductase andO-acetyltransferase (i.e., TA98DNP6, YG1024, and YG1041) as well as standard strains TA 1535, TA1537, TA1538, TA98 and TA100 and strains which provide more information on the base-pair substitution (TA 7001 to 7006). Disperse Blue 291 showed direct-acting mutagenic activity with all strains of Salmonella typhimurium tested, except forTA 1535.According to the classification of Claxton et al. (1991), the potency of this product can be considered moderate (10-100 revertants/µg). In the absence of S9, the nitroreduction is strongly related to the mutagenic activity, because the mutagenicity of Disperse Blue 291 was very low when tested with the strains lacking nitroreductase activity (TA98NR) and was increased with the nitroreductase overproducing strains, (YG1021 and YG1041). The same mutagenic pattern was observed for the acetyltransferase deficient and overproducing strains (TA98DNP6, YG1024, and YG1041) revealing also the importance of the acetyltransferase enzyme in the activation of Disperse Blue 291. Because of the remarkable increase in the response with the nitroreductase and O-acetyltransferase overproducing strain (YG1041), it is likely that the product of the nitroreductase is a substrate for the O-acetyltransferase.

In the presence of S9, the mutagenicity was slightly increased with TA98NR, TA98, YG1021, TA98DNP6, and YG1024 suggesting that, P450 enzymes also have a role in the activation of these compounds, besides the bacterial enzymes. This could be explained by the activation of other radicals of the molecule by the S9 enzymes, for example the —OCH₃ or the —N(CH₂CH₃)₂ of the CI Disperse Blue 291.

 

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

September 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Principles of method if other than guideline:

Due to the positive test result in the Initial Mutation Test (standard plate incorporation method), the preincubation test was not performed.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium:
TA98 hisD3052 Frameshift rfa uvrB +R
TA100 hisG46 Base pair substitution rfa uvrB +R
TA1535 hisG46 Base pair substitution rfa uvrB
TA1537 hisC3076 Frameshift rfa uvrB

Escherichia coli
WP2uvrA trpE65 Base pair substitution uvrA

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

rat S9-Mix

Test concentrations with justification for top dose:

50, 160, 500, 1600, 5000 µg/plate
precipitation from 160 µg/plate onwards

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene, 97.4 % (w/w), Batch number: 39H0945

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

In both tests top agar was prepared which, for the Salmonella strains, contained 100 ml agar (0.6 % (w/v) agar, 0.5 % (w/v) NaC1) with 10 ml of a 0.5 mM histidine-biotin solution.
For E. coli histidine was replaced by tryptophan (2.5 ml, 2.0 mM).
The following ingredients were added (in the following order) to 2 ml of molten top agar at approx. 48 °C:
0.5 ml S9-mix (if required) or buffer
0.1 ml of an overnight nutrient broth culture of the bacterial tester strain
0.1 ml test compound solution (dissolved in DMSO)

Evaluation criteria:

Criteria for a valid assay
The assay is considered valid if the following criteria are met:
— the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
— the positive controls induce increases in the mutation frequency which are significant and within the laboratory's normal range

Criteria for a positive response
A test compound is classified as mutagenic if it has either of the following effects:
a) it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn
If the test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system.

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:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

STERILITY CHECKS AND CONTROL PLATES
Sterility of S9-mix and the test compound were indicated by the absence of contamination on the test material and S9-mix sterility check plates. Control plates (background control and positive controls) gave the expected number of colonies, i.e. values were within the laboratory's historical control range.

SOLUBILITY AND TOXICITY
Disperse Blue 291.1 was dissolved in DMSO and a stock solution of 50 mg/ml was prepared for the highest concentration, which provided a final concentration of 5000 µg/plate. Further dilutions of 1600, 500, 160 and 50 µg/plate were used in the plate incorporation test.
Visible precipitation of Disperse Blue 291.1 on the plates was observed at 160 µg/plate and above.
Disperse Blue 291.1 proved to be not toxic to the bacterial strains.

MUTAGENICITY
In the mutation test Disperse Blue 291.1 was tested for mutagenicity at 5000, 1600, 500, 160 and 50 µg/plate. The number of colonies per plate with each strain as
well as mean values of 3 plates were given.
In the absence of the metabolic activation system the test compound induced a significant and dose-dependent increase in the number of revertant colonies with the bacterial strains TA 1535, TA 1537 and TA 98. In the presence of S9 from rat liver the test compound resulted in relevant increases in the number of revertant colonies with the strains TA 98, TA 100, TA 1535 and TA 1537.
On the basis of these findings a preincubation test is not required according to the guidelines.

Conclusions:

The results lead to the conclusion that Disperse Blue 291:1 is mutagenic in the absence and presence of metabolic activation using the standard Ames Test procedure (plate incorporation test) as described.

Executive summary:

Disperse Blue 291:1 (Batch No. ID-I) was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 98 of Salmonella typhimurium and with Escherichia coli WP2uvrA.

One mutagenicity study was conducted as the standard plate test with the plate incorporation method. The study was performed in the absence and in the presence of a metabolizing system derived from a rat liver homogenate. For the study, the compound was dissolved in DMSO, and each bacterial strain was exposed to five dose levels in the plate incorporation test.

Doses for the plate incorporation test ranged from 50 to 5000 µg/plate.

Control plates without mutagen showed that the number of spontaneous revertant colonies was within the laboratory's historical control. All positive controls gave the expected increase in the number of revertant colonies.

Visible precipitation of Disperse Blue 291:1 on the plates was observed at 160 µg/plate and above.

Toxicity: In the plate incorporation test, toxicity was not observed either with or without metabolic activation.

Mutagenicity: In the absence of a metabolic activation system, the test compound resulted in relevant and dose-dependent increases in the number of revertant colonies with the bacterial strains TA 1535, TA 1537 and TA 98. Also in the presence of rat liver S9-mix, treatment of the cells with Disperse Blue 291.1 resulted in relevant increases in the number of revertant colonies with the bacterial strains TA 100, TA 1535, TA 1537 and TA 98.

Based on these findings a preincubation test is not required according to the guidelines.

Summarizing, it can be stated thatDisperse Blue 291:1 is mutagenicin the standard plate test (plate incorporation test) in the absence and in the presence of a metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test substance did not induce DNA repair (as measured by unscheduled DNA synthesis) in rat liver nor did it induce micronuclei in the polychromatic erythrocytes of treated rats as seen in studies with close structural analogues.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

The test item Disperse Blue 291:1 was tested positivein the Bacteria Reverse Mutation Assay (Ames test), but negative in an in vivo Micronucleus Test in mice, testing for clastogenicity and aneugenicity. Furthermore, a close structural analogue (Disperse Blue 291) was tested positive in an Ames test with nitroreductase and O-acetyltransferase positive Salmonella typhimurium strains, but negative with nitroreductase and O-acetyltransferase negative strains andin a test for unscheduled DNA synthesis. Another structural analogue (SA01) was negative in a mutation assay in mammalian cells. This positive effect in the bacterial mutation assay is a bacteria-specific effect due to bacterial nitro-reductases, which are highly effective in these bacterial strains, but not in mammalian cells.

The nitroreductase family comprises a group of flavin mononucleotide (FMN)- or flavin adenine dinucleotide (FAD) -dependent enzymes that are able to metabolize nitroaromatic and nitroheterocyclic derivatives (nitrosubstituted compounds) using the reducing power of nicotinamide adenine dinucleotide (NAD(P)H). These enzymes can be found in bacterial species and, to a lesser extent, in eukaryotes. The nitroreductase proteins play a central role in the activation of nitro-compounds. Type I nitroreductases can transfer two electrons from NAD(P)H to form the nitroso and hydroxylamino intermediates and finally the amino group. Type II nitroreductases transfer a single electron to the nitro group, forming a nitro anion radical, which in the presence of oxygen generates the superoxide anion in a futile redox cycle, regenerating the nitro group [de Oliveira et al. 2010].

The positive effect in the bacterial reverse mutation test (Ames) was clearly related to a bacteria-specific metabolism of the test substance, as it is well-known for aromatic nitro compounds to be positive in the Ames assay resulting from metabolism by the bacteria-specific enzyme nitro-reductase [Tweats et al. 2012]. This could be also be proved to be true in studies with Disperse Blue 291, which was tested for mutagenic activity in the Salmonella assay with strains with different levels of nitroreductase and O-acetyltransferase[Umbuzeiro et al. 2005]. In this study,Disperse Blue 291 showed mutagenic activity with all standard strains of Salmonella typhimurium tested (TA1537, TA1538, TA98 and TA100), except for TA1535.In nitroreductase and O-acetyltransferasenegative strains (TA98NR, TA98DNP6) not mutagenic activity was observed in the absence of S9, whereas themutagenic activity was increased with the nitroreductaseand/or O‑acetyltransferaseoverproducing strains, (YG1021, YG1024 and YG1041) This shows the importance of the bacterial acetyltransferase enzyme in the activation of Disperse Blue 291. Because of the remarkable increase in the response with the nitroreductase and O‑acetyltransferase overproducing strain (YG1041), it is assumed that the product of the nitroreductaseis a substrate for the O-acetyltransferase. As there was a very slight increase in mutagenicity with TA98NR, TA98, YG1021, TA98DNP6, and YG1024 in the presence of S9, it was assumed that P450 enzymes have also a role in the activation ofDisperse Blue 291, besides the bacterial enzymes. This could however not proven true in studies in mammalian cells (HPRT assay) or in in-vivo studies with Disperse Blue 291 (UDS) or Disperse Blue 291:1 (MNT).

 

It has also been demonstrated in various other publications that this mutagenic activity is a bacteria-specific effect and that these Ames positive nitro-substances are not mutagenic in mammalian assays.

That the reduction of these nitro-compounds to mutagenic metabolites is a bacteria-specific effect is demonstrated in the following by means of the two compounds AMP397 and fexinidazole.

• AMP397 is a drug candidate developed for the oral treatment of epilepsy. The molecule contains an aromatic nitro group, which obviously is a structural alert for mutagenicity. The chemical was mutagenic in Salmonellastrains TA97a, TA98 and TA100, all without S9, but negative in the nitroreductase-deficient strains TA98NR and TA100NR. Accordingly, the ICH standard battery mouse lymphomatkand mouse bone marrow micronucleus tests were negative, although a weak high toxicity-associated genotoxic activity was seen in a micronucleus test inV79 cells [Suter et al. 2002].The amino derivative of AMP397 was not mutagenic in wild type TA98 and TA100. To exclude that a potentially mutagenic metaboliteis released by intestinal bacteria, a Muta^TMMouse study was done in colon and liver with five daily treatments at the MTD, and sampling of 3, 7 and 21 days post-treatment. No evidence of a mutagenic potential was found in colon and liver. Likewise, a comet assay did not detect any genotoxic activity in jejunum and liver of rats, after single treatment with a roughly six times higher dose than the transgenic study, which reflects the higher exposure observed in mice. In addition, a radioactive DNA binding assay in the liver of mice and rats did not find any evidence for DNA binding. Based on these results, it was concluded that AMP397 has no genotoxic potential in vivo. It was hypothesized that the positive Ames test was due to activation by bacterial nitro-reductase, as practically all mammalian assays including fourin vivoassays were negative, and no evidence for activation by mammalian nitro-reductase or other enzymes were seen. Furthermore, no evidence for excretion of metabolites mutagenic for intestinal cells by intestinal bacteria was found.


• Fexinidazolewas in pre-clinical development as a broad-spectrum antiprotozoal drug by the Hoechst AG in the 1970s-1980s, but its clinical development was not pursued. Fexinidazole was rediscovered by the Drugs for Neglected Diseases initiative (DNDi) as drug candidate to cure the parasitic disease human African trypanomiasis (HAT), also known as sleeping sickness. The genotoxicity profile of fexinidazole, a 2-substituted 5-nitroimidazole, and its two active metabolites, the sulfoxide and sulfone derivatives were investigated [Tweats et al. 2012]. All the three compounds are mutagenic in the Salmonella/Ames test; however, mutagenicity is either attenuated or lost in Ames Salmonella strains that lack one or more nitroreductase(s). It is known that these enzymes can nitroreduce compounds with low redox potentials, whereas their mammalian cell counterparts cannot, under normal conditions. Fexinidazole and its metabolites have low redox potentials and all mammalian cell assays to detect genetic toxicity, conducted for this study either in vitro (micronucleus test in human lymphocytes) or in vivo (ex vivo unscheduled DNA synthesis in rats; bone marrow micronucleus test in mice), were negative.

 

Based on these data and the common mechanism between the reduction of these nitro-compounds, which is widely explored in literature [de Oliveira et al. 2010], it is concluded, that the mutagenic effects observed in the Ames test with Disperse Blue 291:1 is a bacteria specific effect and not relevant to mammalians.

Disperse Blue 291:1 and its structural analogues were not genotoxic in the mammalian in-vitro cell mutagenicity test (HPRT assay) and the in-vivo UDS and MNT test. Therefore, a direct genotoxic effect as well as a metabolisation towards genotoxic structures by mammalian species can be excluded.

References

De Oliveira IM, Bonatto D, Pega Henriques JA. Nitroreductases: Enzymes with Environmental Biotechnological and Clinical Importance. InCurrent Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology; Mendez-Vilas, A., Ed.; Formatex: Badajoz, Spain, 2010:1008–1019.

Suter W, Hartmann A, Poetter F, Sagelsdorff P, Hoffmann P, Martus HJ. Genotoxicity assessment of the antiepileptic drug AMP397, an Ames-positive aromatic nitro compound. Mutat Res. 2002 Jul 25;518(2):181-94.

Umbuzeiro GA, Freeman H, Warren SH, Kummrow F, Claxton LD. Mutagenicity evaluation of the commercial product CI Disperse Blue 291 using different protocols of the Salmonella assay. Food and Chemical Toxicology 2005;43:49–56.

Tweats D, Bourdin Trunz B, Torreele E. Genotoxicity profile of fexinidazole--a drug candidate in clinical development for human African trypanomiasis (sleeping sickness). Mutagenesis. 2012 Sep;27(5):523-32.

Additional information

Bacteria reverse mutation

Disperse Blue 291:1 Cl (81.0%purity),was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 98 of Salmonella typhimurium and with Escherichia coli WP2uvrA. One mutagenicity study was conducted as the standard plate test with the plate incorporation method. A preincubation test was not performed, as the plate incorporation test was positive. The study was performed in the absence and in the presence of a metabolizing system derived from a rat liver homogenate. For the study, the compound was dissolved in DMSO, and each bacterial strain was exposed to five dose levels in the plate incorporation test. Doses for the plate incorporation test ranged from 50 to 5000 µg/plate. Control plates without mutagen showed that the number of spontaneous revertant colonies was within the laboratory's historical control. All positive controls gave the expected increase in the number of revertant colonies. Visible precipitation of Disperse Blue 291:1 on the plates was observed at 160 µg/plate and above. Cytotoxicity was not observed in the plate incorporation test, either with or without metabolic activation. In the absence of a metabolic activation system, the test compound resulted in relevant and dose-dependent increases in the number of revertant colonies with the bacterial strains TA 1535, TA 1537 and TA 98. Also in the presence of rat liver S9-mix, treatment of the cells with Disperse Blue 291:1 resulted in relevant increases in the number of revertant colonies with the bacterial strains TA 100, TA 1535, TA 1537 and TA 98. Based on these findings a preincubation test is not required according to the guidelines. Summarizing, it can be stated that Disperse Blue 291:1 is mutagenic in the standard plate test (plate incorporation test) in the absence and in the presence of a metabolic activation system.

Disperse Blue 291 (commercial product; 30-50% dye content) was tested for mutagenic activity in the Salmonella assay with strains with different levels of nitroreductase and O‑acetyltransferase (i.e., TA98DNP6, YG1024, and YG1041) as well as standard strains TA 1535, TA1537, TA1538, TA98 and TA100 and strains which provide more information on the base-pair substitution (TA 7001 to 7006). Disperse Blue 291 showed direct-acting mutagenic activity with all strains ofSalmonella typhimuriumtested, except for TA 1535.According to the classification of Claxton et al. (1991), the potency of this product can be considered moderate (10-100 revertants/µg). In the absence of S9, the nitroreduction is strongly related to the mutagenic activity, because the mutagenicity of Disperse Blue 291 was very low when tested with the strains lacking nitroreductase activity (TA98NR) and was increased with the nitroreductase overproducing strains, (YG1021 and YG1041). The same mutagenic pattern was observed for the acetyltransferase deficient and overproducing strains (TA98DNP6, YG1024, and YG1041) revealing also the importance of the acetyltransferase enzyme in the activation of Disperse Blue 291. Because of the remarkable increase in the response with the nitroreductase and O-acetyltransferase overproducing strain (YG1041), it is likely that the product of the nitroreductase is a substrate for the O-acetyltransferase.

In the presence of S9, the mutagenicity was slightly increased with TA98NR, TA98, YG1021, TA98DNP6, and YG1024 suggesting that, P450 enzymes also have a role in the activation of these compounds, besides the bacterial enzymes. This could be explained by the activation of other radicals of the molecule by the S9 enzymes, for example the —OCH₃ or the —N(CH₂CH₃)₂ of Disperse Blue 291, which however proved not to happen in studied with mammalian cells or in-vivo studies, as shown in the negative results with these studies.

Mammalian cell gene mutation

A study was conducted to determine the genotoxicity of the structural analogue SA01 (90.3% purity) according to OECD Guideline 476.The test item was examined for mutagenic activity by assaying for the induction of 6-thioguanine resistant mutants in Chinese hamster V79 cells after in vitro treatment, both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. Two independent assays were performed using the following dose levels:

Main Assay I (+S9): 600, 400, 267, 178, 119 and 79.0 µg/mL

Main Assay I (-S9): 40.0, 20.0, 10.0, 5.00, 2.50, 1.25 and 0.625 µg/mL

Main Assay II (+S9): 400, 308, 237, 182, 140 and 108 µg/mL

Main Assay II (+S9): 20.0, 15.4, 11.8, 9.10, 7.00 and 5.39 µg/mL

No reproducible five-fold increases in mutant numbers or mutant frequency were observed following treatment with the test item at any dose level, in the absence or presence of S9 metabolism. It is concluded that the substance 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.

Unscheduled DNA Synthesis

Disperse Blue 291 (96%purity)was tested for the ability to induce unscheduled DNA synthesis (UDS) in an in vivo rat hepatocyte assay. Male Fischer 344 rats were treated with a single oral dose of CI Disperse Blue 291 by gavage at 1250, or 2000 mg/kg body weight. The highest test dose, 2000 mg/kg was the limit test dose for a non-toxic test agent in this assay. Animals were killed and hepatocytes prepared four hours and twelve hours following administration of the chemical. Two independent experiments were carried out for each time point. Hepatocytes from treated rats were exposed to [³H]-thymidine and the amount of radioactivity incorporated into the nucleus [N] and an equal area of cytoplasm [C] determined by autoradiography. The cytoplasmic grain count was subtracted from that of the nucleus. The value obtained, the mean net nuclear grain count [N-C], is an index of UDS activity. In the respective testing laboratory, no negative control animal has shown a mean net nuclear grain count greater than zero. An [N-C] of more than zero in a treated animal is therefore considered indicative of a UDS response. Each experiment was validated by concurrent control treatments of rats with corn oil, the solvent for Disperse Blue 291 and with the carcinogens 2-acetylaminofluorene [2AAF] at twelve hours and N-nitrosodimethylamine [NDMA] or 6-p-dimethylaminophenylazobenzthiazole [6BT] at four hours. Solvent treated rats gave rise to mean net grain counts of less than zero, whilst hepatocytes from 2AAF, 6BT or NDMA treated animals had mean net nuclear grain counts of greater than +5. These data showed that background levels of UDS were normal and that the tester animals were responsive to known carcinogens requiring metabolic activation for genotoxic activity. Hepatocytes from Disperse Blue 291 treated animals were assessed for UDS at two dose levels of 1250 and 2000 mg/kg body weight. Treatments with Disperse Blue 291 in no case resulted in a mean net grain count greater than zero, at either time point. It is concluded that, when tested up to a limit dose of 2000 mg/kg body weight, the test sample of Disperse Blue 291 did not induce DNA repair (as measured by unscheduled DNA Synthesis) in hepatocytes from rats treated in vivo.

Mammalian erythrocyte micronucleus test

Rats were treated twice with 2000 mg Disperse Blue 291:1 Cl (81% purity) per kg body weight to study the induction of micronuclei in bone marrow cells. All animals survived after treatment. No signs of toxicity were detected, but all dosed animals showed red discoloured urine 5-6 hours after the administration. The dissection of the animals revealed dark blue coloured contents of the gastro-intestinal tract. The bone marrow smears were examined for the occurrence of micronuclei in red blood cells. The incidence of micronucleated polychromatic erythrocytes in the dose groups with Disperse Blue 291.1 was within the normal range of the negative control groups (mean of micronucleated polychromatic erythrocytes per 2000 cells: 1.7 — 4.9). No statistically significant increase in micronucleated polychromatic erythrocytes was observed. The ratio of polychromatic erythrocytes to total erythrocytes remained essentially unaffected by the test compound and differed less than 20% from the control values. Cyclophosphamide (Endoxan) induced a marked and statistically significant increase in the number of polychromatic erythrocytes with micronuclei, thus indicating the sensitivity of the test system. The results lead to the conclusion that Disperse Blue 291:1 did not cause a substantial increase in micronucleated polychromatic erythrocytes and is not clastogenic in the micronucleus test in vivo under the conditions described in this report.

Justification for classification or non-classification

Based on the results of in vitro and in vivo testing, no classification for genotoxicity is required for the test substance according to CLP (EC 1272/2008) criteria.