Trisodium 7-[[4-chloro-6-[ethyl[3-[[2-(sulphonatooxy)ethyl]sulphonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[(4-methoxy-2-sulphonatophenyl)azo]naphthalene-2-sulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

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:

From November 12, 1999 to December 04, 1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

RA study

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Purity: 52%
Concentration of stock solution in deionized water: 50 mg/mL

Target gene:

Histidine and tryptophan

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: deficient in lipopolysaccharide layer and in DNA excision repair system

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: defective in the uvrA system of DNA repair

Metabolic activation:

with and without

Metabolic activation system:

S-9 fraction (rat liver homogenate)

Test concentrations with justification for top dose:

Plate incorporation test: 1. 0, 50, 160, 500, 1600 and 5000 µg/plate
Repeat plate incorporation test: 2. 0, 50, 160, 500, 1600, 2500 and 5000 µg/plate

Vehicle / solvent:

Deionized water

Untreated negative controls:

yes

Remarks:

untreated controls

Negative solvent / vehicle controls:

yes

Remarks:

deionized water

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 1-methyl-3-nitro-1-nitrosoguanidine and 2-aminoanthracene

Details on test system and experimental conditions:

To 2 mL of molten top agar in a sterile test-tube, were added 0.1 mL of the tester strain culture, graded quantities of the test substance in 0.1 mL solution and, for the S-9 series, 0.5 mL of S-9 Mix. The contents of the test-tube were rapidly mixed and poured onto the surface of previously prepared minimal agar plates with Vogel-Bonner E mixture. The plate were incubated upside down at 37°C for 2 days, after which the number of revertants colonies appearing was counted.

Evaluation criteria:

Doubling of the spontaneous mutation rate (control) and dose-related increase in the mean number of revertants

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

in both tests

Metabolic activation:

with and without

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:

Mutagenicity was negative in all other strains tested.
All positive controls produced significant increases in the number of revertant colonies.

Conclusions:

Under the study conditions, the substance was considered to be mutagenic to Salmonella typhimurium with and without metabolic activation.

Executive summary:

A study was conducted to determine the mutagenic potential of the read-across substance according to OECD Guideline 471, EU Method B13/14 and US EPA OPPTS 870.5100, in compliance with GLP. Four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and Escherichia coli strain WP2uvrA were exposed to the test substance at concentrations of 0 to 5000 µg/plate with or without metabolic activation (S-9 Mix) for 48 h. Negative and positive controls were valid. The test substance induced mutagenic activity in the strain of Salmonella typhimurium TA 1535 both under the presence as well as absence of metabolic activation. The number of revertants was greater than twice the number of spontaneous mutations. Mutagenicity was negative in all other strains tested. Under the study conditions, the substance was considered to be mutagenic with and without metabolic activation (Stammberger, 1999).

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

From November 12, 1999 to December 04, 1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

RA study

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro mammalian chromosomal aberration assay

Specific details on test material used for the study:

Purity: 52%
Concentration of stock solution in MEM Hank's medium: 5 mg/mL

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Liver microsomal fraction (S-9)

Test concentrations with justification for top dose:

Based on preliminary testing for solubility and toxicity (the highest concentration was the highest practicable concentration and it produced a distinct lowering of the mitotic index)
First experiment (3h): 0, 1250, 2500, 3750 and 5000 µg/mL
Second experiment (20h, without S9-mix): 0, 125, 250, 500, 750, 1000, 1500 and 2000 µg/mL (mutagenicity already observed after 3h treatment time)

Vehicle / solvent:

MEM Hank's medium

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

MEM Hank's medium

True negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

2 cultures were used for each concentration.

Rationale for test conditions:

International guidelines and preliminary testing

Evaluation criteria:

A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The number of chromosome aberrations found in the solvent control cultures should reasonably be within the laboratory historical control data range.
b) The positive control substances should produce a statistically significant (Chi-square test, onesided, p < 0.05) increase in the number of cells with chromosome aberrations.
c) A homogeneous response between the replicate cultures is observed.
d) A possible precipitate present on the slides should not interfere with the scoring of chromosome aberrations.

Statistics:

- One-sided Fisher - Exact test
- Criteria for the response:
A test substance was considered positive (mutagenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not mutagenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant increase in the number of cells with chromosome aberrations, both with and without metabolic activation.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

at 3750 and 5000 µg/mL

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

at 2500 µg/mL

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Up to the highest dose, the test subsance induced significant and dose-dependent increases in the number of phases with chromosome aberrations and also in the number of chromosome aberration rates without metabolic activation after 3h treatment time. With metabolic activation, the test substance induced a significant increase in the number of chromosome aberrations exclusive gaps only at a concentration of 2500 µg/mL after 3h treatment time. Although the statistical test did not indicate significance, there was a tendency for higher aberration rates and an increased rate of exchange figures 3h after the start of treatment. This is an indication of heavy chromosomal damage. Based on those results, the longer treatment time (20h) was not necessary;
- Slight microscopical visible precipitation of the test substance was observed at the highest investigated concentrations. Reference mutagens used as positive controls showed significant increase in chromosome aberrations. In the presence and absence of metabolic activation, the test substance was considered to be toxic at the highest dose 5000 µg/mL) to V79 cells after 3h treatment time. In the absence of S9-mix, toxicity was observed at 2000 µg/mL after 20h.

Conclusions:

Under the study conditions, the substance was clastogenic to V79 cells with and without metabolic activation.

Executive summary:

A study was conducted to determine the genotoxic potential of the read-across substance according to OECD Guideline 473, EU Method B10 and US EPA OPPTS 870.5375, in compliance with GLP. Chinese hamster V79 cells were exposed to the test substance at concentrations of 0 to 5000 µg/plate with or without metabolic activation (S-9 Mix) for 3 or 20 h. Slight precipitation of the test substance was observed at the highest investigated concentrations. Reference mutagens used as positive controls showed significant increase in chromosome aberrations. In the presence and absence of metabolic activation, the test substance was considered to be toxic at the highest dose 5000 µg/mL to V79 cells after 3 h treatment. In the absence of S9 -mix, toxicity was observed at 2000 µg/mL after 20 h. Up to the highest dose, the test substance induced significant and dose-dependent increases in the number of phases with chromosome aberrations and also in the number of chromosome aberration rates without metabolic activation after 3 h treatment time. With metabolic activation, the test substance induced a significant increase in the number of chromosome aberrations (exclusive gaps) only at a concentration of 2500 µg/mL after 3 h treatment time. Although the statistical test did not indicate significance, there was also a tendency for higher aberration rates and an increased rate of exchange figures 3 h after the start of treatment. This is an indication of heavy chromosomal damage. Under the study conditions, the substance was clastogenic to V79 cells with and without metabolic activation (Stammberger, 1999).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

From November 12, 1999 to December 04, 1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

RA study

Reason / purpose for cross-reference:

read-across: supporting information

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes

Type of assay:

other: HPRT mutation system in Chinese hamster V79 cells

Specific details on test material used for the study:

Purity: 52%
Solubility: in MEM Hank's medium

Target gene:

enzyme hypoxanthine-guaninphosphoribosyl-transferase (HPRT)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S-9 (from Sprague-Dawley rat liver)

Test concentrations with justification for top dose:

0, 125, 250, 500, 750, 1000, 1750, 2500, 3750 and 5000 µg/mL (depending on preliminary testing for solubility and toxicity)

Vehicle / solvent:

Dilution in MEM Hank's medium

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

MEM Hank's medium

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Mutation assay:
-Treatment of cell cultures. Two experiments 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. Sufficient numbers of 175 cm2 flasks were inoculated with 5.0E05 to 1.0E06 freshly trypsinised V79 cells for culture. For selection of mutants, the medium was supplemented with ca. 11 µg/L thioguanine. All incubations were carried out at ca. 37°C and 4% CO2. Staining was performed with ca. 10% (v/v) methylene blue. Only colonies with more than 50 cells were counted.
Cytotoxicity:
24h after seeding of ca. 4500 cells per well in a microtiter plate, cells were exposed to the test substance for 4h. After the exposure period, the medium was removed, the cells were rinsed twice and stained with crystal violet. Survival plate efficiency) was then determined.

Rationale for test conditions:

Preliminary studies

Evaluation criteria:

The assay is considered valid if:
- The solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency,
- The positive controls induced increases in the mutation frequency which were both statistically significant and within laboratory's normal control range,
- The plating efficiency for the solvent control was greater than 50%.

Statistics:

With the MannWhtney-U-test (compard to negative control data)
The test substance is considered positive (mutagenic) if:
- It reproducibly induces with one of the test substance concentrations a mutation frequency that is three times higher than the spontaneous mutant frequency,
- There is a reproducible dose-related increase in the mutation frequency,
- The survival of the responding dose group is at least 30%.

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary test:
Solubility: only isolated microscopic visible crystals were observed at dose levels of 1000 µg/mL and above.
Toxicity: a toxicity was observed with and without metabolic activation (dose-related manner).
Main test:
Survival: a dose-related decrease was observed at the highest dose of 5000 µg/mL.
Mutation: no relevant reproducible increases in the mutant colonies or mutant frequency which were 3 times higher than the range of the solvent control groups was found. No dose dependency was observed.
The positive and negative control results were valid.

Conclusions:

Under the study conditions, the substance was not mutagenic to V79 cells with and without metabolic activation.

Executive summary:

A study was conducted to determine the mutagenic potential of the read-across substance according to OECD Guideline 476, EU Method B17 and US EPA OPPTS 870.5300, in compliance with GLP. Chinses hamster V79 cells were exposed to the test substance at concentrations of 0 to 5000 µg/plate with or without metabolic activation (S-9 Mix) for 4 h. During preliminary testing, only isolated microscopic visible crystals were observed at dose levels of 1000 µg/mL and above. Toxicity of the test substance was observed with and without metabolic activation in a dose-related manner. The survival of the cells showed a dose-related decrease at the highest dose of 5000 µg/mL. No relevant reproducible increase in the number of mutant colonies or mutant frequency which was 3 times higher than the range of the solvent control groups was found. The positive and negative control results were valid. Under the study conditions, the substance was not mutagenic to V79 cells with and without metabolic activation (Stammberger, 1999).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

From November 12, 1999 to December 04, 1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

RA study

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

other: Mammalian Erythrocyte Micronucleus test

Specific details on test material used for the study:

Purity: 52%
Suspension: in deionized water

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

Origin: Harlan Winkelmann GmbH
Acclimatization: at least 5 days
Body weight (mean): 24.0 and 34.2g (female and male)
Age: ca. 7 weeks
Temperature and relative humidity: 20+/-3°C and 50+/-20%, respectively
Lighting time: 12h daily
Food: ssniff R/M-H (V 1534), ad libitum
Water: tap water, ad libitum

Route of administration:

oral: gavage

Vehicle:

deionized water

Details on exposure:

Twice at an interval of 24h
Dose: 2000 mg/kg bw (based on a preliminary study)
Sacrifice: 24h after administration

Duration of treatment / exposure:

24h

Frequency of treatment:

Twice

Post exposure period:

24h

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

volume: 10 mL/kg bw

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Yes: Endoxan (cyclophosphamide), 50 mg/kg bw administered once orally

Tissues and cell types examined:

Bone marrow from the femora
Erythrocytes

Details of tissue and slide preparation:

A part of the suspension was smeared onto a cleaned slide and air-dried for about 12h. The staining was performed as follows:
5 min in methanol, 5 min in May-Grünwald's solution, brief rinsing in distilled water, 10 min in Giemsa/buffer solution, rinsing in distilled water, drying and coating with Entellan

Evaluation criteria:

Validity assessment and mutagenicity:
2000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. In addition, the ratio (proportion) of polychromatic erythrocytes to 200 total erythrocytes was determined.

Statistics:

One-sided Wilcoxon test
(Both biological and statistical significances)
A substance is considered positive if there is a significant increase in the number of micronucleated polychromatic erythrocytes compared with the concurrent negative control group.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The number of polychromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with the test substance and was not less than 20% of the control value.
Positive control (endoxan): induction of a marked statistically significant increase in the number of polychromatic cells with micronuclei. The ratio of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent.
All animals survived after treatment. No signs of toxicity were observed. The following clinical signs were recorded: red colored urine and faeces.

Conclusions:

Under the study conditions, the substance did not lead to a substantial increase of micronucleated polychromatic erythrocytes and was not clastogenic in the micronucleus test.

Executive summary:

A study was conducted to determine the in vivo clastogenic potential of the read-across substance according to OECD Guideline 474, EU Method B12 and US EPA OPPTS 870.5395, in compliance with GLP. Male and female NMRI mice received by oral gavage the test substance at a concentration of 0 (vehicle alone) or 2000 mg/ kg bw twice at an interval of 24 h. Positive control mice received cyclophosphamide at 50 mg/kg bw. Bone marrow erythrocytes from the femora were analysed. 2000 polychromatic erythrocytes were counted per animal for the presence of micronuclei. In addition, the ratio of polychromatic erythrocytes to 200 total erythrocytes was determined. As a result of the test substance exposure, the number of polychromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with the test substance and was not less than 20% of the control value. The positive control group showed a statistically significant increase in the number of polychromatic cells with micronuclei. All animals survived the treatment and no signs of toxicity were observed. Under the study conditions, the substance did not lead to a substantial increase of micronucleated polychromatic erythrocytes and was not clastogenic in the micronucleus test (Stammberger, 1999).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity, in vitro:

A study was conducted to determine the mutagenic potential of the read-across substance according to OECD Guideline 471, EU Method B13/14 and US EPA OPPTS 870.5100, in compliance with GLP. Four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and Escherichia coli strain WP2uvrA were exposed to the test substance at concentrations of 0 to 5000 µg/plate with or without metabolic activation (S-9 Mix) for 48 h. Negative and positive controls were valid. The test substance induced mutagenic activity in the strain of Salmonella typhimurium TA 1535 both under the presence as well as in the absence of metabolic activation. The number of revertants was greater than twice the number of spontaneous mutations. Mutagenicity was negative in all other strains tested. Under the study conditions, the substance was considered to be mutagenic with and without metabolic activation (Stammberger, 1999).

A study was conducted to determine the mutagenic potential of the substance according to a scientifically acceptable method. Four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and Escherichia coli strain WP2uvrA were exposed to the test substance at concentrations of 0.0 to 5000.0 µg/plate with or without metabolic activation (S-9 Mix from rat liver) for 48 h. Negative (solvent alone) and positive controls were valid. Under the study conditions, the substance was not mutagenic in bacteria with and without metabolic activation (Sumitomo, 1982). The study received a lower Klimisch rating due to poor reporting of study details including some of the important information.

A study was conducted to predict the possible alerts for genotoxicity of the substance using OECD QSAR Toolbox v3.4. Based on the QSAR prediction results, no alerts for genotoxicity was obtained except for micronucleus alerts by ISS were shown by the substance (OECD, 2017).

A study was conducted to determine the genotoxic potential of the read-across substance according to OECD Guideline 473, EU Method B10 and US EPA OPPTS 870.5375, in compliance with GLP. Chinese hamster V79 cells were exposed to the test substance at concentrations of 0 to 5000 µg/plate with or without metabolic activation (S-9 Mix) for 3 or 20 h. Slight precipitation of the test substance was observed at the highest investigated concentrations. Reference mutagens used as positive controls showed significant increase in chromosome aberrations. In the presence and absence of metabolic activation, the test substance was considered to be toxic at the highest dose 5000 µg/mL to V79 cells after 3 h treatment. In the absence of S9 -mix, toxicity was observed at 2000 µg/mL after 20 h. Up to the highest dose, the test substance induced significant and dose-dependent increases in the number of phases with chromosome aberrations and also in the number of chromosome aberration rates without metabolic activation after 3 h treatment time. With metabolic activation, the test substance induced a significant increase in the number of chromosome aberrations (exclusive gaps) only at a concentration of 2500 µg/mL after 3 h treatment time. Although the statistical test did not indicate significance, there was also a tendency for higher aberration rates and an increased rate of exchange figures 3 h after the start of treatment. This is an indication of heavy chromosomal damage. Under the study conditions, the substance was clastogenic to V79 cells with and without metabolic activation (Stammberger, 1999).

A study was conducted to determine the mutagenic potential of the read-across substance according to OECD Guideline 476, EU Method B17 and US EPA OPPTS 870.5300, in compliance with GLP. Chinses hamster V79 cells were exposed to the test substance at concentrations of 0 to 5000 µg/plate with or without metabolic activation (S-9 Mix) for 4 h. During preliminary testing, only isolated microscopic visible crystals were observed at dose levels of 1000 µg/mL and above. Toxicity of the test substance was observed with and without metabolic activation in a dose-related manner. The survival of the cells showed a dose-related decrease at the highest dose of 5000 µg/mL. No relevant reproducible increase in the number of mutant colonies or mutant frequency which was 3 times higher than the range of the solvent control groups was found. The positive and negative control results were valid. Under the study conditions, the substance was not mutagenic to V79 cells with and without metabolic activation (Stammberger, 1999).

Genetic toxicity, in vivo:

A study was conducted to determine the in vivo clastogenic potential of the read-across substance according to OECD Guideline 474, EU Method B12 and US EPA OPPTS 870.5395, in compliance with GLP. Male and female NMRI mice received by oral gavage the test substance at a concentration of 0 (vehicle alone) or 2000 mg/ kg bw twice at an interval of 24 h. Positive control mice received cyclophosphamide at 50 mg/kg bw. Bone marrow erythrocytes from the femora were analysed. 2000 polychromatic erythrocytes were counted per animal for the presence of micronuclei. In addition, the ratio of polychromatic erythrocytes to 200 total erythrocytes was determined. As a result of the test substance exposure, the number of polychromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with the test substance and was not less than 20% of the control value. The positive control group showed a statistically significant increase in the number of polychromatic cells with micronuclei. All animals survived the treatment and no signs of toxicity were observed. Under the study conditions, the substance did not lead to a substantial increase of micronucleated polychromatic erythrocytes and was not clastogenic in the micronucleus test (Stammberger, 1999).

Justification for classification or non-classification

A guideline-compliant Ames study is available for the read-across substance showing positive results. An Ames test was also conducted for the substance itself, showing negative effects. This study was however attributed a lower Klimisch rating due to poor reporting of the study details and missing of some of the important information. In order to resolve the apparent inconsistency in the data, QSAR modelling (through OECD QSAR Toolbox, v3.4) was run for both the substances. There were alerts (DNA binding alerts by OECD) for the read-across substance but not for the substance. However, additional alerts for 'in vivo mutagenicity (micronucleus) by ISS' for both the substances were found. Since the test substance and the read-across substance have similar behaviour in terms of functional groups and the read-across substance represents a worst-case because of the structural alerts for genotoxicity, a weight of evidence approach is proposed. This approach takes into account the (Q)SAR profiling of the test substance, the negative results in the Ames test for the test substnce, the results of genotoxicity studies for the read-across substance and results of literature research for vinyl-sulphone dyes.

It is well known that vinyl-sulphone compounds result in false positive test results in in-vitro tests for clastogenicity (Dearfield KL et al. (1991); Warra TJ et al. (1990)). This is due to the fact that these chemical agents react via the Michael addition reaction. Chemical reactivity via Michael addition is essential for many of the uses for which these compounds are important. As in the currently assessed dye, a structural analogue to Reactive Red 222, vinyl sulphone moieties are used in fibre-reactive dyes (MacGregor et at. (1980)). These compounds are known to deplete glutathione in in vitro test systems, in which the concentration of phase II enzymes is very low. Glutathione plays a role in the detoxification of many compounds. Conjugation with glutathione via Michael addition and subsequent excretion is the most common bio-elimination route for these compounds. Since in-vitro systems have low levels of glutathione, the glutathione depletion leads to a positive result in the in-vitro test system, which is not the case in the in-vivo test system, where glutathione is present in adequate amount, as could be shown in plenty of tests with vinyl sulphone dyes (internal data DyStar). Hence, the in-vivo test produces more reliable data for this kind of substance.

Overall, based on the weight of evidence from in vitro and in vivo genotoxicity studies, the substance is not considered to warrant classification for genotoxicity according to the EU CLP (EC 1272/2008) criteria.