Trisodium 3-[[4-[[6-(anilino)-1-hydroxy-3-sulphonato-2-naphthyl]azo]-5-methoxy-o-tolyl]azo]naphthalene-1,5-disulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an Ames test the substance was tested in Salmonella typhimurium strains TA1535, TA1537, TA98, TA102 and TA100 in presence and absence of metabolic activation. As the substance is an azo-dye the Prival Mitchell modification was included in the test design. No increase in mutant frequency was reported in any of the strains tested both in presence and absence of metabolic activation. The substance is considered to be non-mutagenic under the conditions of this test.

The substance was tested in the L5178Y TK +/- Mouse  Lymphoma Assay according to OECD 490 up to precipitation level. Exposure was either 4 hours (with and without metabolic activation) or 24 hours (without metabolic activation). No increase in the mutant frequency at the TK +/- locus was reported (evaluation with the Global Evaluation Factor (GEF) of 126 E-06). Therefore it is concluded that the substance is non-mutagenic in this assay.

The substance was tested in an in vitro micronucleus test (OECD 487). Human lymphocytes were exposed for 4 hours (with and without metabolic activation) or 24 hours (without metabolic activation). After the exposure cytokinesis was inhibited by treatment with Cytochalasin B. There was no inhibition of the CBPI and the concentrations to be evaluated were limited by precipitate at 128 ug/mL. No increase in the percentage of micronucleated binuclear cells was observed in any of the treatments. Therefore it can be concluded that the substance is not clastogenic.

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:

19 October 2016 to 25 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Principles of method if other than guideline:

includes the Prival and Mitchell (1982) modification to assess the mutagenic activity of azo dyes.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

the concentrations were corrected for 8.6% water

Target gene:

histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: University of California, Berkeley, on culture discs, on 04 August 1995
- Methods for maintenance in cell culture if applicable: stored at -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34

Metabolic activation:

with and without

Metabolic activation system:

test 1 : phenobarbital/beta-naphthaflavone induced rat liver S9; Test 2: uninduced hamster liver S9

Test concentrations with justification for top dose:

Test 1 (pre-incubation with rat S-9): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Test 2 (pre-incubation with hamster S9): 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: substance fully soluble up to 50 mg/L

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

congo red

mitomycin C

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 30 min at 37 ± 3 °C
- Exposure duration: 48 h at 37 ± 3 °C

NUMBER OF REPLICATIONS: 3/concentration

DETERMINATION OF CYTOTOXICITY
- Method: visible reduction in the growth of the bacterial background lawn

Evaluation criteria:

a substance is considered positive when:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester
strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).

Statistics:

Dunnetts Regression Analysis

Key result

Species / strain:

other: TA1535, TA1537, TA98, TA102 and TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The mean positive control value of Congo Red used in conjunction with TA100 in the second mutation test (Privall Mitchell modification) showed an 1.9-fold ncrease over the concurrent vehicle control. This was considered acceptable as the test concentrations were all within 84 to 125% of vehicle controls (without dose response).

Conclusions:

Based on the findings in this test the substance is considered non-mutagenic in bacteria

Executive summary:

In an Ames test the substance was tested in salmonella typhimurium strains TA1535, TA1537, TA98, TA102 and TA100 in presence and absence of metabolic activation. As the substance is an azo-dye the Prival Mitchell modification was included in the test design. No increase in mutant frequency was reported in any of the strains tested both in presence and absence of metabolic activation. The substance is considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 October 2016 to 5 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

NA

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: blood of healthy non-snoking volunteers
- Cell cycle length: 16 hours
- Sex, age and number of blood donors: Preliminary Toxicity Test: male, aged 28 years
Main Experiment: male, aged 25 years
- Cell type: lymphocytes of fresh heparinized whole blood (stimulated to divide by PHA)

CULTURE MEDIA USED: Whole blood cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 inhumidified air.

Culture medium lymphocytes:
8.05-9.05 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized whole blood

Cytokinesis block (if used):

Cytochalasin B added to block actin polymerisation

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/beta-naphthaflavone induced rat liver S9

Test concentrations with justification for top dose:

4-hour without S9 0*, 8, 16, 32*, 64*, 128*, 256, MMC 0.2*
4-hour with S9 (2%) 0*, 8, 16, 32*, 64*, 128*, 256, CP 5*
24-hour without S9 0*, 8, 16, 32*, 64*, 128*, 256, DC 0.075*

* evaluated concentration

Concentrations were corrected for purity based on water content

Vehicle / solvent:

Minimal Essential Medium (MEM)

Untreated negative controls:

yes

Remarks:

MEM

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcine

Details on test system and experimental conditions:

Cells were exposed to the substance in MEM in presence and/or absence of S9 for 4 or 24 hours at approximately 37 ºC. Thereafter the cultures were centrifuged, the treatment medium was replaced with original culture medium, supplemented with Cytochalasin B at a final concentration of 4.5 µg/mL, and then incubated for a further 24 hours.Thereafter cells were fixed with methanol/glacial acetic acid (19:1 v/v) and stored at 4 ºC prior to slide making. Slides were stained with Giemsa and dried. Thereafter cells (500 per culture) were scored for mono-, bi- and multinuclei and CPBI (plus cytostasis). In the main experiment 1000 cells per culture (2 cultures per concentration) were also scored for the number of micronucleated cells (and the number of micronuclei per cell)

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: 2000 binucleated cells was analyzed per concentration (1000 binucleated cells per culture, two cultures per concentration). Cells with 1, 2 or more micronuclei were recorded as such but the primary analysis was on the combined data. The criteria for identifying micronuclei were that they were round or oval in shape, non-refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.

DETERMINATION OF CYTOTOXICITY: 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI (Cytokinesis Block Proliferation Index) value expressed as a percentage of the vehicle controls. Cytostasis was calculated from these values

Evaluation criteria:

Negative when:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase.
3. The results in all evaluated dose groups should be within the range of the laboratory historical control data.

Positive when:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is an increase which can be considered to be dose-related.
3. The results are substantially outside the range of the laboratory historical negative control data.

Statistics:

Chi-squared Test

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

concentrations limited by precipitate at 128 ug/mL

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes:

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

concentrations limited by precipitate at 128 ug/mL

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: 7.26-7.35
- Effects of osmolality: 267-284 mOsm

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: available in the report
- Negative (solvent/vehicle) historical control data: available in the report

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI

Remarks on result:

other: 4 h exposure

4-h without metabolic activation

Dose Level (µg/mL)	Mean CBPI	% Cytostasis	Mean % Binucleated cells with MN
0	1.49	0	0.10
32 M	1.53	0	0.30
64 M	1.55	0	0.65
128 M P	1.54	0	0.10
MMC 0.2	1.48	2	6.25***

 

4-h with metabolic activation

Dose Level (µg/mL)	Mean CBPI	% Cytostasis	Mean % Binucleated cells with MN
0	1.79	0	0.55
32 M	1.77	3	0.05
64 M	1.81	0	0.45
128 M P	1.70	11	0.20
CP 5	1.28	65	6.20***

 

24-h without metabolic activation

Dose Level (µg/mL)	Mean CBPI	% Cytostasis	Mean % Binucleated cells with MN
0	1.97	0	0.40
32 M	1.98	0	0.35
64 M	1.95	2	0.10
128 M P	1.95	2	0.45
DC 0.075	1.81	16	3.45***

 

M=purple coloration of cultures without cells

P=precipitate

*** p <0.001

Conclusions:

In the in vitro micronucleus assay the substance did not show clastogenic effects

Executive summary:

The substance was tested in an in vitro micronucleus test (OECD 487). Human lymphocytes were exposed for 4 hours (with and without metabolic activation) or 24 hours (without metabolic activation). After the exposure cytokinesis was inhibited by treatment with Cytochalasin B. There was no inhibition of the CBPI and the concentrations to be evaluated were limited by precipitate at 128 ug/mL. No increase in the percentage of micronucleated binuclear cells was observed in any of the treatments. Therefore it can be concluded that the substance is not clastogenic.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 October 2016 to 23 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

``Kanpoan No. 287 - - Environment Protection Agency``
``Eisei No. 127 - - Ministry of Health and Welfare``
``Heisei 09/10/31 Kikyoku No. 2 - - Ministry of International Trade & Industry``

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro gene mutation study in mammalian cells

Target gene:

TK

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED: L5178Y TK+/- 3.7.2c mouse lymphoma cell line
- Source of cells: MRC Cell Mutation Unit at the University of Sussex, Brighton, UK
- Cell cycle length:ca 12 hours
- Methods for maintenance in cell culture if applicable: stored in liquid nitrogen at approximately -196°C

MEDIA USED
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes, before freezing

Additional strain / cell type characteristics:

other: TK+/-

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/beta-naphthaflavone induced rat liver S9

Test concentrations with justification for top dose:

4 h exposure: 0.98, 1.95, 3.91, 7.81, 15.63, 31.25 µg/mL
24 h exposure: 1.95, 3.91, 7.81, 15.63, 31.25, 62.5 µg/mL

Top dose based on presence of precipitate

Vehicle / solvent:

RPMI 1640 medium (R0)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

A pretest was conducted on cell cultures at 5 x 10 E5 cells/mL, using a 4-hour exposure period both with and without metabolic activation (S9), and at 1.5 x 10E5 cells/mL using a 24-hour exposure period without S9. The dose range was set at 7.81 to 2000 µg/mL. After exposure cells were washed twice, resuspended and counted with a Coulter counter. The cultures were serially diluted to 2 x 10E5 cells/mL. and incubated at 37 ˚C with 5% CO2 in air and sub-cultured after 24 hours. After a further 24 hours the cultures were counted to assess Suspension Growth (SG)

In the main study exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) in R0 or R10 medium (total volume to 20 mL). The solutions were incubated at 37°C for 4 or 24 hours with continuous shaking. the initial cell number was 1 x 10E6 cells/mL for 4 hour exposures and 0.3 x 10E6 cells/mL for 24 hour exposure.
At the end of the treatment period, for each experiment, the cells were washed twice, resuspended in R20 medium and incubated at 37°C with 5% CO2 (subcultured and counted every 24 hours -> Relative Suspension Growth (%RSG)) for 2 days.

On Day 2 of the experiment, the cells were counted again, diluted to 10E4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5-trifluorothymidine (TFT) in 96-well microtitre plates. These plates were scored after incubation at 37°C with 5% CO2 for the presence of large and small colonies. On day additional cells were diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.

Statistics:

Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al., 1989).

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

concentrations tested based on presence of precipitate

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

concentrations tested based on presence of precipitate

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no (7.26-7.35)
- Effects of osmolality: no (267-284 mOs)
- Precipitation:
main study: at 15.63 µg/mL in the 4-hour cultures and at and above 62.5 µg/mL in the 24-hour culture

RANGE-FINDING/SCREENING STUDIES:
4 h without S9: no cytotoxicity, precipitate at at 125 µg/mL
4 h with S9: cytotoxicity at 500 µg/mL, precipitate at at 125 µg/mL
24 h without S9: cytotoxicity at 2000 µg/mL, precipitate at at 62.5 µg/mL

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%) are reported in an annex to the report

Remarks on result:

other: 4 hour exposure

Treatment (µg/ml)	4-hours -S-9 Treatment					Treatment (µg/ml)	4-hours +S-9 Treatment					Treatment (µg/ml)		24-hours -S-9
		%RSG	RTG	MF§				%RSG	RTG	MF§				%RSG	RTG	MF§
0		100	1.00	122.55		0		100	1.00	130.11		0		100	1.00	134.46
0.98		93	0.85	135.55		0.98		108	1.07	133.94		0.49	Ø	104
1.95		94	0.81	145.91		1.95		106	1.10	117.83		0.98	Ø	102
3.91		95	0.95	140.28		3.91		117	1.09	134.18		1.95		107	1.02	135.55
7.81		89	0.83	129.81		7.81		110	0.95	136.45		3.91		109	1.20	115.63
15.63		85	0.86	136.57		15.63		104	1.03	121.91		7.81		106	1.23	126.98
31.25		82	0.71	171.61*		31.25		119	1.11	133.55		15.63		88	1.04	135.64
62.5	Ø	92				62.5	Ø	111				31.25		87	1.09	147.15
125	Ø	95				125	Ø	97				62.5		79	0.96	154.75
	Linear	trend		*			Linear	trend		NS			Linear	trend		NS
EMS						CP						EMS
400		68	0.44	1415.62		1.5		76	0.53	928.69		150		40	0.37	1527.79

 § Positive wells per tray, 96 wells plated

 * P<0.05

 

Conclusions:

The substance is considered non-mutagenic in the L5178Y TK +/- Mouse Lymphoma Assay

Executive summary:

The substance was tested in the L5178Y TK +/- Mouse  Lymphoma Assay according to OECD 490 up to precipitation level. Exposure was either 4 hours (with and without metabolic activation) or 24 hours (without metabolic activation). No increase in the mutant frequency at the TK +/- locus was reported (evaluation with the Global Evaluation Factor (GEF) of 126 E-06). Therefore it is concluded that the substance is non-mutagenic in this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the outcome of the available in vitro tests, the substance does not need to be classified for mutagenicity according to Regulation (EC) No 1272/2008 (CLP).