p-[4,5-dihydro-4-[[2-methoxy-5-methyl-4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulphonic acid, sodium salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was not detected to show signs for genetic toxicity in in-vitro tests for mutagenicity and cytogenicity, neither in presence nor in absence of metabolic activation.

In three Ames tests, there was once one slightly higher reverse mutation rate observed for the strain TA100 with metabolic activation with rat liver S9. However, this results was not seen in the Prival modification and was not seen in both other Ames tests and is hence considered to be an outlier which is normal for repeated biological tests. All other types of cells did not give an indication on a genetically toxic effect. In conclusion, the test item does not show adverse effects on mutagenicity or cytogenicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine (S. typhimurum) and tryptophane (E. coli)

Species / strain / cell type:

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

Remarks:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of Sprague-Dawley rat liver homogenate

Test concentrations with justification for top dose:

Test concentrations: 0, 4, 20, 100, 500, 2500, 10000 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

methylmethanesulfonate

other: 2-aminoanthracene, methylhydrazone derivate, streptocotocine, ENNG

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 4

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:

DETERMINATION OF CYTOTOXICITY
- Any supplementary information relevant to cytotoxicity: substance is not cyctotoxic up to 10 mg/plate

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

The test substance was not found to be mutagenic in any strain of Salmonella typhimurum (TA 1535, TA 1537, TA 38 TA 100, TA 98) or Escherichia coli (WP2 uvr A), neither with nor without metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1988-11-22 to 1988-12-08

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

only Salmonella typhimurium strains TA 100, TA 1535, TA 1537 and TA 98 were used

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 from rat or hamster liver homogenate

Test concentrations with justification for top dose:

Highest test concentration of 10 mg/L derived from cytotoxicity test which showed no toxic effects up to 10 mg/L.
Test concentration: 0, 4, 20, 100, 500, 2500, 10000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: H2O bidest.
- Justification for choice of solvent/vehicle: water solubility 2 g/L

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 72 h
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): 10 mL histidine biotin solution (0.5 M)

NUMBER OF REPLICATIONS: 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Increase of revertant colonies with metabolic activation.

Conclusions:

The test substance was found to be slightly mutagenic in the presence of metabolic activation for one strain of Salmonella typhimurum (TA100) but not without metabolic activation. The test substance was not found to be mutagenic in both presence and absence of metabolic activation for three other bacteria strains (TA 1535, 1537 and TA 98).

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1989-09-27 to 1989-10-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

only Salmonella typhimurium strains TA 100, TA 1535, TA 1537 and TA 98 were used; E. coli was covered in former studies

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

E. coli WP2 uvr A

Remarks:

has already been tested in study 02/82

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 from rat or hamster liver homogenate

Test concentrations with justification for top dose:

Highest test concentration of 5 mg/L derived from cytotoxicity test which showed no toxic effects up to 5 mg/L.
Test concentrations: 0, 4, 20, 100, 500, 2500, 10000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (0.1 mL)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 72 h
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): 10 mL histidine biotin solution (0.5 M)

NUMBER OF REPLICATIONS: 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

The test substance was not found to be mutagenic in any strain of Salmonella typhimurum (TA 1535, TA 1537, TA 100, TA 98), neither with nor without metabolic activation.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988-09-07 to 1988-12-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1983

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Laboratory of Genetic Toxicology of Hoechst AG, Germany
- Suitability of cells: used for many years, high proliferation rate, recommended by guideline
- Methods for maintenance in cell culture if applicable: 37°C in 25 cm² plastic flasks, seeding with 1-3 x 10^5 cells in 5 mL MEM medium with 10% fetal calf serum, subcultured twice a week

MEDIA USED
- Properly maintained: yes
- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 from male Sprague-Dawley rat

Test concentrations with justification for top dose:

Test concentrations: 0, 0 (solvent control), 50, 100, 200. 300, 500, 750, 1000, 1500, 2000 µg/L
Cytotoxic effects at 1500 µg/L and 2000 µg/L in absence of metabolic activation, no cytotoxic effects up to 2000 µg/l with metabolic activation system.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: H2O bidest.
- Justification for choice of solvent/vehicle: water soluble

Untreated negative controls:

yes

Remarks:

completely untreated

Negative solvent / vehicle controls:

yes

Remarks:

treated with solvent (water)

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Two days old exponentially growing stock cultures which were over 50 % confluent were trypsinised and a single cell suspension was prepared. The trypsin concentration was 0.5 % in Ca-Mg-free salt solution. 1-2 x 10^6 cells/flask were seeded into four 80 cm² plastic flasks containing 15 mL MEM with 10 % FCS (6 h preparation). 4-6 x 10^5 cells/flask were seeded into four 25 cm² plastic flasks containing 5 mL MEM with 10 % FCS (18 h preparation). 2-4 x 10^5 cells/flask were seeded into four 25 cm² plastic flasks containing 5 mL MEM with 10 % FCS (28 h preparation).

After 24 h the medium was replaced with medium containing 5 % FCS and the test substance, both without S9-mix and with 40 µL/mL S9-mix.
After 2 h this medium was replaced with normal medium after rinsing once with physiological saline solution.

Treatment was performed with 3 concentrations of the test substance.

Evaluation criteria:

Chromosome abberations: changes resulting from damage expressed in bothe sister chromatids at the same locus
Chromatid abberations: changes resulting from damage expressed as breakage of a single chromatid or breakage and/or reunion between chromatids
Numerical abberations: variations of the normal chromosome number

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

The test item does not induce chromosome mutations (abberations) in V79 Chinese hamster cells, neither in presence nor in absence of metabolic activations.

Executive summary:

The test substance was examined for mutagenic activity in V79 Chinese hamster cells. The induction of chromosome aberrations after in vitro treatment was investigated in the presence and absence of a fraction of liver homogenate for metabolic activation (S9-mix).

A preliminary cytotoxicity experiment was performed in order to select appropri­ate dose levels for the mutagenicity study. The test substance produced a signi­ficant cytotoxic effect (reduction of plating efficiency) without metabolic activation from 1500 µg/ml up to the limit of solubility (2000 µg/ml). No cyto-toxic effect was observed with metabolic activation up to the limit of solub­ility. For mutagenicity testing two independent cell cultures with and without metabolic activation (S9-mix) up to the limit of solubility (2000 ug/ml) were used.

For main experiment dose levels of 200, 600 and 2000 µg/ml were used, in the absence and in the presence of S9-mix metabolic activation.

The test compound did not induce a significant increase in the number of chromo­some aberrations at any preparation time and dose level of the test substance. No relevant cytotoxic effect (reduction of mitotic index) of the compound was observed in the main experiments. Marked increases in the rate of chromosome aberrations were obtained with the positive control substances indicating the sensitivity of the assay.

In conclusion, the test substance does not induce chromosome mutations (=aberrations) in V79 Chinese hamster cells, neither in the presence nor in the absence of a metabolic activation system, under the experimental conditions described.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 2019- February 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

The mutation assay method used in this study is based on the identification of L5178Y colonies which have become resistant to a toxic thymidine analogue trifluorothymidine (TFT).This analogue can be metabolised by the enzyme thymidine kinase (TK) into nucleosides,
which are used in nucleic acid synthesis resulting in the death of TK-competent cells.TK-deficient cells, which are presumed to arise through mutations in the TK gene, cannot metabolise trifluorothymidine and thus survive and grow in its presence. In the L5178Y mouse lymphoma cells, the gene which codes for the TK enzyme is located on chromosome 11. Cells which are heterozygous at the TK locus (TK+/−) may undergo a single step forward mutation to the TK−/− genotype in which little or no TK activity remains. The cells used, L5178Y TK+/−, are derived from one of the two clones originated from a thymic tumour induced in a DBA/2 mouse by methylcholanthrene. The use of the TK mutation system in L5178Y mouse lymphoma cells has been well characterised and validated (D. Clive
et al., 1979) and is accepted by most of the regulatory authorities. The mouse lymphoma assay often produces a bimodal size distribution of TFT resistant
colonies designated as small or large. It has been evaluated that point mutations and deletions within the active allele (intragenic event) produce large colonies. Small colonies result in part from lesions that affect not only the active TK allele but also a flanking gene whose expression modulates the growth rate of cells.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI medium supplemented with Horse serum.
- Properly maintained: yes; Permanent stocks of the L5178Y TK+/− cells are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use.
- Periodically checked for Mycoplasma contamination: yes
- The generation time and mutation rates (spontaneous and induced) have been checked in this laboratory.
- Prior to use, cells were cleansed of pre-existing mutants.

Metabolic activation:

with and without

Metabolic activation system:

S9 tissue fraction: Species: Rat; Strain: Sprague Dawley; Tissue: Liver Inducing Agents: Phenobarbital – 5,6-Benzoflavone Producer: MOLTOX, Molecular Toxicology, Inc. Batch Number: 3971

Test concentrations with justification for top dose:

The test item was found to be soluble at the maximum concentration of 20.0 mg/mL. On the basis of this result a concentration of 2000 µg/mL (the upper
limit to be tested stated in the Study Protocol) was selected as the top dose level to be used in the cytotoxicity test.

Based on the results obtained in the preliminary trial, three independent assays for mutation at the TK locus were performed using the following dose levels:
Main Assay I (+S9, 3 hour treatment): 2000, 1000, 500, 250 and 125 ug/mL.
Main Assay I (-S9, 3 hour treatment): 2000, 1000, 500, 250 and 125 ug/mL.

Since the results obtained precluded making a conclusion of positive or negative results, an additional experiment (Main Assay II) was performed in the absence and presence of S9 metabolic activation, using the short treatment time and the following concentrations:
Main Assay II (+S9, 3 hour treatment): 2000, 1670, 1390, 1160, 579 and 289 ug/mL.
Main Assay II (-S9, 3 hour treatment): 2000, 1250, 781, 488, 244, 122 and 61.0 ug/mL.

Negative results were obtained inMain Assay II, thus a third experiment (Main Assay III) in the absence of S9 metabolism was performed, using a longer treatment time (24 hours) and the dose levels described in the following table:
Main Assay III (-S9, 24 hour treatment): 500, 400, 320, 256, 128, 64.0 and 32.0 ug/mL.

Vehicle / solvent:

Test item solutions were prepared using complete medium (RPMI 1640 Minimal medium).

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

Cytotoxicity assay
A preliminary cytotoxicity test was performed in order to select appropriate dose levels for the mutation assays. In this test a wide range of dose levels of the test item was used and the survival of the cells was subsequently determined. Treatments were performed in the absence and presence of S9 metabolic activation for3 hours and for 24 hours only in the absence of S9 metabolic activation. A single culture was used at each test point. After washing in Phosphate Buffered Saline (PBS), cells were resuspended in 20mL of complete medium (10%). Cell concentrations were adjusted to 8 cells/mL using complete medium (20%) and, for each dose level, 0.2mL was plated into 96 microtitre wells. The plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 7-10 days. Wells containing viable clones were identified by eye using background illumination and then counted.

Mutation assay
The mutation assay was performed including vehicle 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. In the first experiment (Main Assay I), the cells were exposed to the test item for a short treatment time (3 hours). Since result obtained precluded making a conclusion of positive or negative results, an additional experiment (Main Assay II) was performed in the absence and presence of S9 metabolic activation, using the short treatment time and a different
range of concentrations. Clear negative results were obtained, thus a third experiment (Main Assay III) in the absence of S9 metabolism was performed, using a longer treatment time (24 hours). After washing in Phosphate Buffered Saline (PBS), cells were resuspended in fresh complete medium (10%) and cell densities were determined. The number of cells was adjusted to give 2×105 cells/mL. The cultures were incubated at 37°C in a 5% CO2 atmosphere (100%
nominal relative humidity) to allow for expression of the mutant phenotype.
During the expression period (two days after treatment), the cell populations were subcultured in order to maintain them in exponential growth. At the end of this period, the cell densities of each culture were determined and adjusted to give 2×105 cells/mL. After dilution, the cell suspensions in complete medium B (20%) were supplemented with trifluorothymidine (final concentration 3.0 µg/mL) and an estimated 2 × 103 cells were plated in each well of four 96-well plates.
Plateswere incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified by eye using background illumination and counted. In addition, the number of wells containing large colonies as well as the number of those containing small colonies were scored.
After dilution, in complete medium A (20%), an estimated 1.6 cellmaking a conclusion of positive or negative results, an additional experiment (Main Assay II) was performed in the absence and presence of S9 metabolic activation, using the short treatment time and a different range of concentrations.s/wellwere plated in eachwell of two 96-well plates. These plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified as above and counted.

Evaluation criteria:

For a test item to be considered mutagenic in this assay, it is required that:
1. The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell method (126×10^−6) at one or more doses.
2. There is a significant dose-relationship as indicated by the linear trend analysis.
Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance. Any increase in mutant frequency should lie outside the historical control range to have biological relevance.

Statistics:

Statistical analysis was performed according to UKEMS guidelines (RobinsonW.D., 1990).

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Survival after treatment
In the first experiment, in the absence of S9 metabolic activation, severe toxicity, reducing relative total growth (RTG) to 1 and 6% of the concurrent negative control value, was observed at 2000 and 1000 µg/mL, respectively. Moderate toxicity was noticed at the next lower concentration, reducing RTG to 23% of the negative control, while mild toxicity (RTG=50%) was seen at 250 µg/mL. No relevant toxicity was noted at the lowest concentration tested.
In the presence of S9 metabolism, treatment with the test item at 2000 µg/mL yielded marked toxicity reducing RTG to 10% of the concurrent negative control value, moderate toxicity (RTG=39%) was noted at the next lower concentration, while no relevant toxicity was observed over the remaining concentrations tested.
In the second experiment, in the absence of S9 metabolism, dose relate toxicity was noticed from 122 µg/mL onwards, reducing RTG to 15 and 12% of the concurrent negative control at 781 and 1250 µg/mL, respectively. In the presence of S9 metabolic activation, severe toxicity was observed at the two highest dose levels of 2000 and 1670 µg/mL (RTG lower than 10%), while treatment at 1390 µg/mL yielded moderate toxicity reducing RTG 21%. Dose related toxicity was seen at the next two lower concentrations.In the third experiment, in the absence of S9 metabolic activation using a long treatment time, the highest dose level selected (500 µg/mL) yielded severe toxicity reducing RTG to 6% of the concurrent negative control value. At the next two lower dose levels of 400
and 320 µg/mL toxicity from marked to moderate was noticed and RTG was reduced to 14 and 25%, respectively. Mild toxicity was seen over the remaining dose levels tested. At low survival levels, the mutation data are prone to a variety of artefacts (selection effects, sampling error, founder effects). Mechanisms other than direct genotoxicity per se can lead to positive results that are related to cytotoxicity and not genotoxicity (e.g. events associated with apoptosis, endonuclease release fromlysosomes, etc.). For this reason it is generally recommended that such data are treated with caution or excluded from consideration. Accordingly, we have excluded from the statistical analyses, mutation data obtained at the following concentration/treatment condition combinations:
Main Assay I −S9 3h 2000
Main Assay II −S9 3h 2000
Main Assay II +S9 3h 2000 and 1670
Main Assay III −S9 24h 500

Mutation results
InMain Assay I, no relevant increase in mutant frequencies was observed following treatment with the test item, in the absence of S9 metabolism. However, the number of analysable concentration was not sufficient for a clear evaluation of test item mutagenicity, thus an additional experiment was performed. In the presence of S9 metabolism, a statistically significant increase inMutant Frequency (MF) was seen at the highest concentration of 2000 µg/mL where the RTG was reduced to 10%. A linear trend was indicated and the observed increase was higher than the Global Evaluation Factor (GEF). However, a result
would not be considered positive if the increase in MF occurred only at or below 10% RTG; hence an additional experiment was performed to assess mutagenicity at appropriately toxic concentrations. In Main Assay II, in the absence of S9 metabolism, statistically significant increases in mutant frequency were observed at the two highest analysable concentrations and a linear trend was indicated. However, the IMF values were lower than the GEF and the mutation
frequencies fell within the normal range (50−170×10−6 viable cells) for negative controls. In addition, these increases were observed only at high levels of cytotoxicity (RTG values between 10-20 %), thus the result obtained was not considered of biological relevance. In the presence of S9 metabolism, no relevant increase in mutant frequencies was observed at any analysable concentration.
In Main Assay III a statistically significant increase in MF was noticed only at the highest analysable concentration of 400 µg/mL, which elicits a high level of cytotoxicity (RTG=14%). Moreover, this increase was lower than the GEF and the mutation frequency fell within the normal range (50−170×10−6 viable cells) for negative controls, thus the result obtained in this treatment series was considered of no biological relevance.

Conclusions:

REACTIVE YELLOW 15 does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Executive summary:

The test item REACTIVE YELLOW15 was examined for mutagenic activity by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using afluctuation method.

A preliminary solubility trial indicated that the maximum practicable concentration of the test item in the final treatment medium was 2000 µg/mL using RPMI 1640 minimal medium as solvent. On the basis of this result, a cytotoxicity assay was performed. Both in the absence and presence of S9 metabolic activation, the test item was assayed at a maximum dose level of 2000 µg/mL and at a wide range of lower dose levels: 1000, 500, 250,

125, 62.5, 31.3, 15.6 and 7.81 µg/mL. In the absence of S9 metabolic activation, using the 3 hour treatment time, moderate toxicity was observed at the highest concentration tested, reducing relative survival (RS) to 32% of the concurrent negative control value; slight toxicity was seen at the next lower concentration of 1000 µg/mL (RS= 66%). No relevant toxicity was noted over the remaining concentrations tested. Using the 24 hour treatment time, no cells survived treatment at the two highest concentrations tested, marked toxicity was noted at the next lower concentration (RS=12%), while mild toxicity was observed at 250 µg/mL, reducing relative survival to 52%. Slight or no relevant toxicity was observed over the remaining dose levels tested. Following treatment in the presence of S9 metabolic activation, using the short treatment time (3 hours), test item treatment at 2000 µg/mL yielded mild toxicity reducing

RS to 56%, while no relevant toxicity was observed over the remaining dose levels tested. Based on the results obtained in the preliminary trial, the assay for mutation at the TK locus was performed using the dose levels described in the following table:

Main Assay I −S9 3h 2000, 1000, 500, 250 and 125 µg/mL

Main Assay I +S9 3h 2000, 1000, 500, 250 and 125 µg/mL

In the mutation assay in the absence of S9 metabolism, severe toxicity was observed at the two highest concentrations, reducing Relative Total Growth (RTG) to 1 and 6%. The next lower dose level of 500 µg/mL yielded moderate toxicity, reducing RTG to 23%, while mild or no relevant toxicity was seen at the two remaining concentrations tested. In the presence of S9 metabolic activation, marked toxicity (RTG=10%) was observed at the highest

concentration tested, while moderate reduction of RTG (39%) was seen at the next lower dose level of 1000 µg/mL. No relevant toxicity was noticed at the remaining concentrations tested.

No relevant increase in mutant frequencies was observed following treatment with the test item, in the absence of S9 metabolism. However, the number of analysable concentration was not sufficient for a clear evaluation of test item mutagenicity, thus an additional experiment was performed. In the presence of S9 metabolism, a statistically significant increase in Mutant Frequency (MF) was seen at the highest concentration of 2000 µg/mL where the RTG was reduced to 10% and a linear trend was also indicated. The observed increase was higher than the Global Evaluation Factor (GEF). However, since a result would not be considered positive if the increase in MF occurred only at or below 10% RTG, an additional experiment was deemed necessary to assess mutagenicity at appropriately toxic concentrations. Based on the results obtained, an additional assay for mutation (Main Assay II) was performed,

both in the absence and presence of S9 metabolism, using the short treatment and the following dose levels:

Main Assay II −S9 3h 2000, 1250, 781, 488, 244, 122 and 61.0 µg/mL

Main Assay II +S9 3h 2000, 1670, 1390, 1160, 579 and 289 µg/mL

Adequate levels of cytotoxicity, covering a range from the maximum to slight or no toxicity, were observed in both treatment series. In the absence of S9 metabolism, statistically significant increases in mutant frequency

were observed at the two highest analysable concentrations and a linear trend was also indicated. However, these increases were observed only at RTG levels between 10-20 % and the InducedMutant Frequencies (IMF) were lower that the GEF. In the presence of S9 metabolism, no relevant increase in mutant frequencies was observed at any analysable concentration.

Since negative results were obtained inMain Assay II, a third experiment in the absence of S9 metabolism (Main Assay III) was performed using a longer treatment time (24 hours) and the following concentrations:

Main Assay III −S9 24h 500, 400, 320, 256, 128, 64.0 and 32.0 µg/mL

Adequate levels of cytotoxicity, covering a range from the maximum (RTG: 10-20%) to slight or no toxicity (RTG: 60-100%), were observed. A statistically significant increase in MF was noticed only at the highest analysable concentration of 400 µg/mL, which elicits a high level of cytotoxicity (RTG 14%). Moreover, this increase was lower than the GEF and thus was not considered a positive result.

Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system.

It is concluded that REACTIVE YELLOW 15 does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Reactive Yellow 15 was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium. The mutagenicity studies were conducted in the standard plate test (Ames Test) and in a modified preincubation test (Prival Test). The studies were performed in the absence and in the presence of a metabolizing system derived from rat or hamster liver homogenate. A dose range of 6 different doses from 4 µg/plate to 5000 µg/plate was used. Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive con­trol compounds gave the expected increase in the number of revertant colonies.

Toxicity: The test compound proved to be not toxic to the bacterial strains. 5000 µg/plate was chosen as top dose level for the mutagenicity study.

Ames Test: In the absence of the metabolic activation system the test com­pound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of a metabolic activation system, treatment of the cells with Reactive Yellow 15 did not result in relevant increases in the number of revertant colonies.

Summarizing, it can be stated that Reactive Yellow 15 is not mutagenic in these bacterial test systems either with or without exogenous metabolic activation at the dose levels investigated.

Prival Test: In the presence of hamster liver S-9 Mix (30 %) using the preincubation method according to Prival Reactive Yellow 15 did not induce a significant increase in the number of revertant colonies, with any of the tester strains. Also in the presence of rat liver S-9 Mix (30 %) Reactive Yellow 15 did not induce a significant increase in the number of revertant colonies.

Summarizing, it can be stated that Reactive Yellow 15 is not mutagenic in the presence of 30 % rat liver S-9 Mix and the standard plate test (Ames Test) and in the presence of 30 % Syrien golden hamster liver S-9 Mix and the preincubation method according to Prival.

Furthermore,

Reactive Yellow 15 was tested for mutagenicity with the strains Salmonella typhimurium TA98, TA100, TA1S35, TA1S37, TA1538 and Escherichia coli WP2 uvrA at concentrations of 4 µg to 10000 µg/plate. Under the conditions we employed, Reactive Yellow 15 in concentrations of 4 µg to 10000 µg/plate showed no mutagenic activity.

The test item was examined for mutagenic activity by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using afluctuation method.

A preliminary solubility trial indicated that the maximum practicable concentration of the test item in the final treatment medium was 2000 µg/mL using RPMI 1640 minimal medium as solvent. On the basis of this result, a cytotoxicity assay was performed. Both in the absence and presence of S9 metabolic activation, the test item was assayed at a maximum dose level of 2000 µg/mL and at a wide range of lower dose levels: 1000, 500, 250,

125, 62.5, 31.3, 15.6 and 7.81 µg/mL. In the absence of S9 metabolic activation, using the 3 hour treatment time, moderate toxicity was observed at the highest concentration tested, reducing relative survival (RS) to 32% of the concurrent negative control value; slight toxicity was seen at the next lower concentration of 1000 µg/mL (RS= 66%). No relevant toxicity was noted over the remaining concentrations tested. Using the 24 hour treatment time, no cells survived treatment at the two highest concentrations tested, marked toxicity was noted at the next lower concentration (RS=12%), while mild toxicity was observed at 250 µg/mL, reducing relative survival to 52%. Slight or no relevant toxicity was observed over the remaining dose levels tested. Following treatment in the presence of S9 metabolic activation, using the short treatment time (3 hours), test item treatment at 2000 µg/mL yielded mild toxicity reducing

RS to 56%, while no relevant toxicity was observed over the remaining dose levels tested. Based on the results obtained in the preliminary trial, the assay for mutation at the TK locus was performed using the dose levels described in the following table:

Main Assay I −S9 3h 2000, 1000, 500, 250 and 125 µg/mL

Main Assay I +S9 3h 2000, 1000, 500, 250 and 125 µg/mL

In the mutation assay in the absence of S9 metabolism, severe toxicity was observed at the two highest concentrations, reducing Relative Total Growth (RTG) to 1 and 6%. The next lower dose level of 500 µg/mL yielded moderate toxicity, reducing RTG to 23%, while mild or no relevant toxicity was seen at the two remaining concentrations tested. In the presence of S9 metabolic activation, marked toxicity (RTG=10%) was observed at the highest

concentration tested, while moderate reduction of RTG (39%) was seen at the next lower dose level of 1000 µg/mL. No relevant toxicity was noticed at the remaining concentrations tested.

No relevant increase in mutant frequencies was observed following treatment with the test item, in the absence of S9 metabolism. However, the number of analysable concentration was not sufficient for a clear evaluation of test item mutagenicity, thus an additional experiment was performed. In the presence of S9 metabolism, a statistically significant increase in Mutant Frequency (MF) was seen at the highest concentration of 2000 µg/mL where the RTG was reduced to 10% and a linear trend was also indicated. The observed increase was higher than the Global Evaluation Factor (GEF). However, since a result would not be considered positive if the increase in MF occurred only at or below 10% RTG, an additional experiment was deemed necessary to assess mutagenicity at appropriately toxic concentrations. Based on the results obtained, an additional assay for mutation (Main Assay II) was performed,

both in the absence and presence of S9 metabolism, using the short treatment and the following dose levels:

Main Assay II −S9 3h 2000, 1250, 781, 488, 244, 122 and 61.0 µg/mL

Main Assay II +S9 3h 2000, 1670, 1390, 1160, 579 and 289 µg/mL

Adequate levels of cytotoxicity, covering a range from the maximum to slight or no toxicity, were observed in both treatment series. In the absence of S9 metabolism, statistically significant increases in mutant frequency

were observed at the two highest analysable concentrations and a linear trend was also indicated. However, these increases were observed only at RTG levels between 10-20 % and the InducedMutant Frequencies (IMF) were lower that the GEF. In the presence of S9 metabolism, no relevant increase in mutant frequencies was observed at any analysable concentration.

Since negative results were obtained inMain Assay II, a third experiment in the absence of S9 metabolism (Main Assay III) was performed using a longer treatment time (24 hours) and the following concentrations:

Main Assay III −S9 24h 500, 400, 320, 256, 128, 64.0 and 32.0 µg/mL

Adequate levels of cytotoxicity, covering a range from the maximum (RTG: 10-20%) to slight or no toxicity (RTG: 60-100%), were observed. A statistically significant increase in MF was noticed only at the highest analysable concentration of 400 µg/mL, which elicits a high level of cytotoxicity (RTG 14%). Moreover, this increase was lower than the GEF and thus was not considered a positive result.

Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system.

It is concluded that the test item does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

 

 

The test substance was examined for cytogenic activity in V79 Chinese hamster cells. The induction of chromosome aberrations after in vitro treatment was investigated in the presence and absence of a fraction of liver homogenate for metabolic activation (S9-mix).

A preliminary cytotoxicity experiment was performed in order to select appropri­ate dose levels for the mutagenicity study. The test substance produced a signi­ficant cytotoxic effect (reduction of plating efficiency) without metabolic activation from 1500 µg/ml up to the limit of solubility (2000 µg/ml). No cyto-toxic effect was observed with metabolic activation up to the limit of solub­ility. For mutagenicity testing two independent cell cultures with and without metabolic activation (S9-mix) up to the limit of solubility (2000 ug/ml) were used.

For main experiment dose levels of 200, 600 and 2000 µg/ml were used, in the absence and in the presence of S9-mix metabolic activation.

The test compound did not induce a significant increase in the number of chromo­some aberrations at any preparation time and dose level of the test substance. No relevant cytotoxic effect (reduction of mitotic index) of the compound was observed in the main experiments. Marked increases in the rate of chromosome aberrations were obtained with the positive control substances indicating the sensitivity of the assay.

In conclusion, the test substance does not induce chromosome mutations (=aberrations) in V79 Chinese hamster cells, neither in the presence nor in the absence of a metabolic activation system, under the experimental conditions described.