Reaction mass of tetrasodium 7,7'-(carbonyldiimino)bis[4-hydroxy-3-[(2-methyl-4-sulphonatophenyl)azo]naphthalene-2-sulphonate] and tetrasodium 4-[[1-hydroxy-6-[[[[5-hydroxy-6-[(2-methyl-4-sulphonatophenyl)azo]-7-sulphonato-2-naphthyl]amino]carbonyl]amino]-3-sulphonato-2-naphthyl]azo]benzoate and tetrasodium 4,4'-[carbonylbis[imino(1-hydroxy-3-sulphonatonaphthalene-6,2-diyl)azo]]dibenzoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

2012, from 10 January to 06 March

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

refer to section 13 of IUCLID

IUPAC Name:

refer to section 13 of IUCLID

Details on test material:

Please refer to confidential details on test material

Method

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Cells: are characterised by their high proliferation rate (10-l2 h doubling time of the BSL BIOSERVICE stock cultures) and their cloning efficiency, usually more than 50%. The cells obtain a near diploid karyotype (40 1 2 chromosomes). They are heterozygous at the Thymidine Kinase (TK) locus in order to
detect mutation events at the TK- locus. To prevent high backgrounds arising from spontaneous mutation, cells lacking TK can be eliminated by culturing cells in RPMI 1640 supplemented with: 9.0 µg/mL hypoxanthine; 15.0 µg/mL thymidine; 22.5 µg/mL glycine; 0.1 µg/mL methotrexate
The cells are resuspended in medium without methotrexate but thymidine, hypoxanthine and glycine for l-3 days.
- St6orage: large stock cultmes of the cleansed L5l78Y cell line are stored over liquid nitrogen (vapour phase) in the cell bank of BSL BIOSERVICE.
- Periodically checked: each cell batch is routinely checked for mycoplasma infection.
Thawed stock cultures are maintained in plastic culture flasks in RPMI l640 complete medium and subcultured three times per week.

Mammalian Microsomal Fraction S9 Homogenate
The S9 liver microsomal fraction was prepared at BSL BIOSERVICE GmbH. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and B-naphthotlavone (100 mg/kg bw) for three consecutive days by oral route.
The following quality control determinations were performed:
a) Biological activity in:
· the Salmonella typhimurium assay using 2-aminoanthracene
- the mouse lymphoma assay using benzo[a]pyrene
- the chromosome aberration assay using cyclophosphamide.
b) Sterility Test
A stock of the supernatant containing the microsomes was frozen in aliquots of 2 and 4.5 mL and stored at ≤ -75°C.
The protein concentration in the S9 preparation (Lot: 2411 ll) was 39 mg/mL.

S9 mix:
An appropriate quantity of the S9 supematant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the following concentrations: 8 mM MgCl2, 33 mM KCI, 5 mM Glucose—6·phosphate
5 mM NADP in 100 mM sodium-phosphate-buffer pH 7.4. During the experiment the S9 mix was stored on ice.

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9 Homogenate

Test concentrations with justification for top dose:

Experiment I, with and without metabolic activation:
10, 50, 100, 200, 400, 600, 1800 and 5000 µg/mL
Experiment II, with metabolic activation:
30, 70, 150, 350, 700, 2100, 3600 and 5000 µg,/mL
and without metabolic activation:
10, 20, 50, 100, 200, 600, 1200 and 2500 µ/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no vehicle
- Justification for choice of solvent/vehicle: A solubility test was performed with different solvents and vehicles up to the maximum recommended concentration of 5 mg/mL. The test item was not soluble in any of the investigated vehicles. Therefore the test item was suspended in cell culture medium (RPMI + HS).

Controls

Controls 1

Untreated negative controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Controls 2

Untreated negative controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation

Controls 3

Untreated negative controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With metabolic activation

Details on test system and experimental conditions:

PRE-EXPERIMENTAL for TOXICITY
The toxicity of the test item was determined in pre-experiments up to a maximum concentration of 5 mg/mL. For pre-experiment l six concentrations [156, 313, 625, 1250, 2500, 5000 µg/mL] were tested with and without metabolic activation. For pre-experiment ll without metabolic activation (24 h long·term exposure) six concentrations [10, 100, 300, 800, 2000, 5000 µg/mL] were tested. The experimental conditions in these pre—experiments were the same as described below in the paragraph experimental performance.

METHOD OF APPLICATION: RPMI medium; short-term experiment: 11mL with 5% horse serum; long-term experiment: 25mL with 7.5% horse serum

DURATION
Short-term
- Exposure duration: 4 hours
- Removal of test item: by centrifugation; 200 x g, 10 min
- Expression time : 2 days at 37°C in 5% CO2/95% humidified air
- Cell density: detrmined each day and adjusted to 3x10^5 cell/mL in a total culture volume of 20 mL, if necessary
- Determination of cells number: by automated cell counting
Long-term
- Exposure duration: 24 hours
- Removal of test item: by centrifugation; 200 x g, 10 min
- Expression time : 2 days at 37°C in 5% CO2/95% humidified air
- Cell density: detrmined each day and adjusted to 3x10^5 cell/mL in a total culture volume of 20 mL, if necessary
- Determination of cells number: by automated cell counting

DETERMINATION of CLONING EFFICIENCY (CE)
Detrmined by seeding a statistical number of 1.6 cell/well in two 96-well plates
- Incubation: Cells were incubated for at least 6 days at 37°C in a humidified atmosphere with 5% CO2
- Selective medium: additionally, cultures were seeded in selective medium. Cells from each experimental group were seeded in four 96-well plates at a density of approximately 2000 cells/well in 200 µL selective medium with TFT. The plates were scored after an incubation period of -14 days at 37 °C in 5% CO2/95% humidified air.
- Mutant frequency determination: calculated dividing the number of TFT resistant colonies by number of cells plated for selection, corrected for the plating efficiency of cells from the same culture grown in the absence of TFT

SUSPENSION GROWTH (SG)
Reflects the number of times the cell number increases from the starting cell density. The relative total growth (RTG) is the product of the relative suspension growth (RSG calculated by comparing the SG of the dose groups with the SG of the control) and the relative cloning efficiency (RCE) for each culture: RTG = RSG x RCE /100. The mutant frequencies obtained from the experiments are compared with the Global Evaluation Factor (GEF). To arrive at a GEF was analyzed distributions of negative/vehicle mutant frequencies of the MLA. The GEF is defined as the mean of the negative/vehicle mutant frequency plus one standard deviation. The non-parametric Mann-Whitney test is applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls.

POSITIVE CONTROLS
Without metabolic activation
- Name: EMS; Ethylmethanesulfonate
- Supplier: Sigma
- Catalogue No.: M 0880
- Batch No.: BCBF0736V and BCBGl395V, respectively
- Dissolved in: Medium
- Final concentrations: 200 ug/mL and 300 ug/mL

- Name: MMS; Methylmethanesulfonate
- Supplier: Sigma
- Catalogue No.: M 4016
- Batch No.: 87596LJ
- Dissolved in: 0.9% NaCl
- Final concentration: 10 ug/mL

With metabolic activation
- Name: B[a]P, Benzo[a]pyrene
- Supplier: Sigma
- Catalogue No.: B 1 760
- Batch No.: 097Kl293
- Dissolved in: DMSO, Dimcthylsulfoxide; final concentration in RPMI medium 1%
- Final concentration: 2.5 µg/mL

The dilutions of the stock solutions of the positive controls were prepared on the day of the experiment and used immediately.
The stability of the positive control substances in solution is proven by the mutagenic response in the expected range.

ACCEPTABILITY OF THE ASSAY
A mutation assay is considered acceptable if it meets the criteria mentioned in current international guidelines and the current recommendations of the IWGT (l l, 12, 13, 14, 1 5):
- At least three out of four 96-well plates from the TFT resistance-testing portion of the experiment are scorable.
- The cloning efficiency of the negative and/or solvent controls is in the range 65% -120%.
- The spontaneous mutant frequency in the negative and/or solvent controls is in the range 50-170 per 10 cells
- The cell number of the negative/solvent controls should undergo 8-32 fold increase during a 2 day growth period (short—term treatment) or 32-180 fold increase during a 3 day growth period (long—term treatment).
- The clastogenic positive controls (MMS and B[a]P) have to produce an induced mutant frequency (total mutant frequency minus concurrent negative control mutant frequency) of at least 300 per 106 cells with at least 40% of the colonies being small colonies or with an induced small colony mutant frequency of at least 150 per 106 cells The RTG must be greater than 10%.

Evaluation criteria:

The test item is considered mutagenic if following criteria are met (13, 14, 15):
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 per 106 cells
- A dose-dependent increase in mutant frequency is detected.
Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (240% of total colonies) is an indication for potential
clastogenic effects and/or chromosomal aberrations.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend test is negative.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Growth inhibition was observed without metabolic activation.

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: in experiment I with and without metabolic activation precipitation of the test item was noted from concentrations of 50 µg/rnL and higher; in experiment II with metabolic activation precipitation was noted from concentrations of 30 µg/mL and higher; in experiment II without metabolic activation from concentrations of 10 µg/mL and higher.

In experiment 1 with metabolic activation all validity criteria were met.
The negative controls showed mutant frequencies within the acceptance range of 50-170 mutants/10^6 cells, according to the IWGT criteria. The mutant frequencies of the negative controls were 123.3 and 128.8 mutants/10^6 cells, the positive control B[a]P induced a distinct increase in mutant frequency with 627.7 mutants/10^6 cells. The mutant frequencies induced by the test item did not show a biologically relevant increase. The GEF of 126 was not exceeded in any of the dose groups showing induced mutant values between -5.6 and 69.1 mutants/10^6 cells. A statistical analysis confirmed that most of the mutant values were significantly different to the mutant frequencies of the negative controls, but there was no evidence for a dose-response relationship.

ln experiment 1 without metabolic activation all validity criteria were met.
The negative controls showed mutant frequencies within the acceptance range of 50-170 mutants/10^6 cells, according to the IWGT criteria. The mutant frequencies of the negative controls were 135.2 and 163.3 mutants/10^6 cells, the positive controls EMS and MMS induced a distinct increase in mutant frequency with 625.8 and 529.7 mutants/10^6 cells. The mutant frequencies induced by the test item did not show a biologically relevant increase. The GEF of 126 was not exceeded in any of the dose groups showing induced mutant frequencies between -61.5 and 68.5 mutants/10^6 cells. A statistical analysis continned that most of the mutant frequencies were significantly different to the mutant frequencies of the negative controls, but there was no evidence for a dose-response relationship.

ln experiment 2 with metabolic activation all validity criteria were met.
The negative controls showed mutant frequencies within the acceptance range of 50-170 mutants/10^6 cells, according to the IWGT criteria. The mutant frequencies of the negative controls were 149.0 and 150.1 mutants/10^6 cells, the positive control B[a]P induced a distinct increase in mutant frequency with 696.9 mutants/10^6 cells. The mutant frequencies induced by the test item did not show a biologically relevant increase. The GEF of 126 was not exceeded in any of the dose groups showing induced mutant frequencies between -32.6 and 34.2 mutants/10^6 cells. None of the observed mutant frequencies were statistically significantly increased over those from the negative controls.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In the described mutagenicity test under the experimental conditions reported, the test item Direct Red 23 is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5l78Y cells.

Executive summary:

The test item Direct Red 23 was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The selection of the concentrations used in the main experiments was based on data from the pre-experiments. In experiment I 5000 µg/mL (with and without metabolic activation) was selected as the highest concentration. In experiment ll 5000 µg/mL (with metabolic activation) and 2500 µg/mL (without metabolic activation) were selected as the highest concentrations. Experiment 1 with and without metabolic activation and experiment ll with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as a 24 h long-term exposure assay.

The test item was investigated at the following concentrations:

- Experiment I

with and without metabolic activation:

10, 50, 100, 200, 400, 600, 1800 and 5000 µg/mL

- Experiment ll

with metabolic activation:

30, 70, 150, 350, 700, 2100, 3600 and 5000 µg/mL

and without metabolic activation:

10, 20, 50, 100, 200, 600, 1200 and 2500 µg/mL

Precipitation of the test item was noted in all experiments.

Growth inhibition was observed in experiment I and ll without metabolic activation.

ln experiment I with metabolic activation the relative total growth (RTG) was 93.7% for the highest concentration (5000 µg/mL) evaluated. The highest concentration evaluated without metabolic activation was 5000 µg/mL with a RTG of 65.0%.

In experiment II with metabolic activation the relative total growth (RTG) was 71.5% for the highest concentration (5000 µg/mL) evaluated. The highest concentration evaluated without metabolic activation was 2500 µg/mL with a RTG of 12.9%.

In experiment I and ll no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). The Global Evaluation Factor (GEF; defined as the mean of the negative/vehicle mutant frequency

plus one standard deviation; data gathered from ten laboratories [13, 14, 15]) was not exceeded by the induced mutant frequency at any concentration.

No dose-response relationship was observed.

Additionally, in experiment I and II colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (with and without metabolic activation).

EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.

Conclusion

ln conclusion, in the described mutagenicity test under the experimental conditions reported, the test item Direct Red 23 is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5l78Y cells.