Xanthylium, 9-(2-carboxyphenyl)-3,6-bis(diethylamino)-, 4-[(5-chloro-2-hydroxyphenyl)azo]-4,5-dihydro-3-methyl-1-phenyl-3H-pyrazol-3-one 4,5-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-3-methyl-1-phenyl-3H-pyrazol-3-one 3-[[1-[[(2-ethylhexyl)amino]carbonyl]-2-oxopropyl]azo]-2-hydroxy-5-nitrobenzoate cobaltate complexes

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Xanthylium, 9-(2-carboxyphenyl)-3,6-bis(diethylamino)-, 4-[(5-chloro-2-hydroxyphenyl)azo]-4,5-dihydro-3-methyl-1-phenyl-3H-pyrazol-3-one 4,5-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-3-methyl-1-phenyl-3H-pyrazol-3-one 3-[[1-[[(2-ethylhexyl)amino]carbonyl]-2-oxopropyl]azo]-2-hydroxy-5-nitrobenzoate cobaltate complexes

EC Number:

276-160-2

EC Name:

Xanthylium, 9-(2-carboxyphenyl)-3,6-bis(diethylamino)-, 4-[(5-chloro-2-hydroxyphenyl)azo]-4,5-dihydro-3-methyl-1-phenyl-3H-pyrazol-3-one 4,5-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-3-methyl-1-phenyl-3H-pyrazol-3-one 3-[[1-[[(2-ethylhexyl)amino]carbonyl]-2-oxopropyl]azo]-2-hydroxy-5-nitrobenzoate cobaltate complexes

Cas Number:

71888-93-2

Molecular formula:

C63H66ClN10O12xCo

IUPAC Name:

Xanthylium, 9-(2-carboxyphenyl)-3,6-bis(diethylamino)-, 4-[(5-chloro-2-hydroxyphenyl)azo]-4,5-dihydro-3-methyl-1-phenyl-3H-pyrazol-3-one 4,5-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-3-methyl-1-phenyl-3H-pyrazol-3-one 3-[[1-[[(2-ethylhexyl)amino]carbonyl]-2-oxopropyl]azo]-2-hydroxy-5-nitrobenzoate cobaltate complexes

Constituent 2

Reference substance name:

Xanthylium,9-(2-carboxyphenyl)-3,6-bis(diethylamino)-,4-[(5-chloro-2-hydroxyphenyl)azo]-4,5-dihydro-3-methyl-1-phenyl-3H-pyrazol-3-one 4,5-dihydro-4-[(2-hydroxy-5-nitrophenyl)-azo]3-methyl-1-phenyl-3H-pyrazol-3-one 3-[[1-[[(2-ethyl-hexyl)amino]carbonyl]-2-oxopropyl]azo]-2-hydroxy-5-nitrobenzoate cobaltate complexes

IUPAC Name:

Xanthylium,9-(2-carboxyphenyl)-3,6-bis(diethylamino)-,4-[(5-chloro-2-hydroxyphenyl)azo]-4,5-dihydro-3-methyl-1-phenyl-3H-pyrazol-3-one 4,5-dihydro-4-[(2-hydroxy-5-nitrophenyl)-azo]3-methyl-1-phenyl-3H-pyrazol-3-one 3-[[1-[[(2-ethyl-hexyl)amino]carbonyl]-2-oxopropyl]azo]-2-hydroxy-5-nitrobenzoate cobaltate complexes

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Batch Manufactured By: Clariant

Supplied By: Clariant India Private Limited, BUPI, Product Stewardship-India, Reliable Tech Park, Gut No. 31, Village Elthan, Thane-Belapur Road, Airoli, Navi Mumbai 400708, India

Test Item: Savinyl-Feuerrot 3GLS (Savinyl Fire Red 3GLS)

Synonym: Solvent Red 124 (S.R.124)

CAS No.: 71888-93-2

Physical Appearance: Red powder

Purity as per CoA: 98.8% (w/w) mixture of two comp.1-3 with cobalt (Co3+) as anion and comp. 4 as cation

Batch No.: AAM0021703

Manufactured Date: Unknown

Expiry Date: 16 January 2022

Recommended Storage Condition: Ambient (+15 to +25°C)

pH: about 5

Density: 1.18 g/cm3

Molecular weight: 1154 - 1342 g/mol

Method

Target gene:

This mammalian cell mutation assay system detects point mutations involving base substitutions, deletions, frameshifts and rearrangements within the locus. The enzyme hypoxanthine guanine phosphoribosyl transferase (hprt) catalyses phosphorylation of purines in one of the purine salvage pathways. The selective agent used in this assay, 6-Thioguanine (6-TG), is also a substrate for this enzyme and cells that retain the functional hprt enzyme are susceptible to the cytotoxic effects of 6-TG. Forward mutations that result in the loss of the functional hprt gene render cells resistant to 6-TG. These mutant cells can be quantitated after an expression period by cloning in culture medium supplemented with 6-TG, the selective agent.

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 homogenate

Test concentrations with justification for top dose:

A) 4 B) 15 C) 58 and D) 230 µg/mL (factor of 4)

Vehicle / solvent:

DMSO was used as the vehicle control.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Details on test system and experimental conditions:

- Source of the Test System:
American Type Culture Collection, P. O. Box 1549, Manassas, VA 20108, USA

- Storage of Test System
Stock cultures of the CHO-K1 cell line were stored in the test facility as frozen permanents in liquid nitrogen.

Evaluation criteria:

- Evaluation and Interpretation : When all the validity criteria are fulfilled:

a.A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:

•At least one of the test concentrations exhibits a statistically significant increase in number of aberrations compared with the concurrent vehicle control
•The increase is dose-dependent when evaluated with an appropriate trend test
•Any of the results are outside the distribution of the historical vehicle control data

b.A test chemical is considered to be clearly negative if, in all experimental conditions examined:

•None of the test concentrations exhibits a statistically significant increase in number of aberrations compared with the concurrent vehicle control
•There is no concentration-related increase when evaluated with an appropriate trend test
•All results are inside the distribution of the historical vehicle control data

c.The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.

Statistics:

A power transformation procedure (Snee and Irr, 1981) was used with which, the observed mutant frequency was transformed.
Statistical analysis of the experimental data was carried out using validated copies of SYSTAT Statistical package version 12.0. In cases where analysis of variance was significant at p < 0.05, a Dunnett’s test was conducted, comparing each treatment group and the positive control to the vehicle control (p < 0.05).

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

TABLE 3.         Determination of pH of Test Medium

Treatment (mg/mL)	pH at the beginning of exposure to treatment		pH at the end of exposure to treatment
	With S9	Without S9	With S9	Without S9
DMSO	7.10	7.03	7.00	7.20
4	7.07	7.10	7.09	7.15
8	7.11	7.12	7.10	7.13
20	7.15	7.09	7.00	7.14
50	7.19	7.14	7.12	7.10
128	7.20	7.18	7.15	7.07
320	7.15	7.21	7.13	7.09
800	7.13	7.20	7.12	7.10
2000	7.21	7.18	7.15	7.12

 

Note: The test item precipitated in the test medium at and above 800 µg/mL both in the presence and absence of metabolic activation at 3 hours post-treatment.

TABLE 2.         Determination of Osmolality of Test Medium

Treatment (mg/mL)	Osmolality at the beginning of exposure to treatment (OSMOL/kg)		Osmolality at the end of exposure to treatment (OSMOL/kg)
	With S9	Without S9	With S9	Without S9
DMSO	0.470	0.463	0.483	0.456
320	-	-	0.471	0.452
800	-	-	0.459	0.455
2000	0.439	0.440	0.460	0.434

 

TABLE 3.         Results of Preliminary Cytotoxicity Test

Treatment (mg/mL)	3-hour exposure with metabolic activation				3-hour exposure without metabolic activation
	Cloning Efficiency (CE)	Cells at end of treatment (x10^5/flask)	Adjusted Cloning Efficiency (ACE)	Relative Survival (%RS)	Cloning Efficiency (CE)	Cells at end of treatment (x10^5/flask)	Adjusted Cloning Efficiency (ACE)	Relative Survival (%RS)
DMSO	0.93	14.70	0.83	100	0.94	14.05	0.80	100
4	0.88	14.05	0.75	90	0.86	13.40	0.70	88
8	0.66	13.70	0.55	66	0.82	11.85	0.59	74
20	0.63	11.00	0.42	51	0.67	11.10	0.45	56
50	0.60	9.85	0.36	43	0.61	10.50	0.39	49
128	0.58	7.35	0.26	31	0.48	9.00	0.26	33
320	0.10	6.25	0.04	5	0.09	6.85	0.04	5
800	No colony growth				No colony growth
2000

Note: Baseline cell count (No. of cells at the beginning of treatment): 16.5 x 10⁵cells/mL                      

          CE and ACE values are rounded of 2 decimal points and RS values are rounded off to the nearest whole number

TABLE 4.         Parallel Cytotoxicity Test Results from Experiment 1

Treatment µg/mL	No. of Colonies /Flask			CE*	ACE	RS %
	1	2	3
DMSO	191	182	190	0.95	0.93	100
	196	184	193
4	180	175	170	0.88	0.81	87
	178	181	172
15	131	128	130	0.67	0.56	60
	141	137	132
58	117	120	115	0.59	0.40	43
	122	112	114
230	60	65	62	0.32	0.18	19
	71	59	61
3-MCA	152	150	147	0.77	0.55	59
	157	161	152

TABLE 5.         Parallel Cytotoxicity Test Results from Experiment 2

Treatment µg/mL	No. of Colonies /Flask			CE*	ACE	RS %
	1	2	3
DMSO	184	192	190	0.95	0.95	100
	182	195	198
4	170	184	169	0.88	0.79	83
	179	182	164
15	141	137	140	0.69	0.56	59
	132	145	128
58	110	118	112	0.58	0.40	42
	117	120	115
230	61	65	75	0.34	0.19	20
	78	64	60

TABLE 6.         Summary Results of the Gene Mutation Assay in the Presence of Metabolic Activation (Experiment 1)

Treatment µg/mL	Mutation Assay Flasks						Cloning Efficiency of Mutant Colonies	Cloning Efficiency Flasks				6-TG Mutants per 10^6Clonable Cells (MF)
	TG Colonies/Flask							No. of Colonies/Flask
	1	2	3	4	5	Total		1	2	3	CE*
DMSO	3	0	3	1	2	17	0.0000085	189	191	184	0.92	9.24
	1	3	1	1	2			178	175	185
4	3	1	1	0	2	16	0.000008	171	182	176	0.89	8.99
	3	2	2	1	1			182	180	174
15	0	2	3	2	3	21	0.000011	170	168	184	0.86	12.21
	3	2	0	4	2			182	167	165
58	3	1	3	1	1	21	0.000011	180	171	174	0.85	12.35
	2	1	3	5	1			172	161	160
230	4	1	2	0	2	18	0.000009	158	160	157	0.80	11.25
	2	2	3	2	0			164	155	161
3-MCA	37	34	36	35	28	340	0.000170	147	156	155	0.77	220.78
	36	30	38	32	34			153	160	157

TABLE 7.         Summary Results of the Gene Mutation Assay in the Absence of Metabolic Activation (Experiment 2)

Treatment µg/mL	Mutation Assay Flasks						Cloning Efficiency of Mutant Colonies	Cloning Efficiency Flasks				6-TG Mutants per 10^6Clonable Cells (MF)
	TG Colonies/Flask							No. of Colonies/Flask
	1	2	3	4	5	Total		1	2	3	CE*
DMSO	2	3	2	5	0	22	0.0000110	190	187	191	0.94	11.70
	2	3	2	3	0			185	183	186
4	2	2	1	2	3	22	0.000011	177	180	174	0.88	12.50
	2	3	2	2	3			184	170	172
15	3	2	2	1	1	16	0.000008	167	179	165	0.87	9.20
	2	2	1	1	1			178	181	177
58	0	3	2	1	4	22	0.000011	160	172	158	0.83	13.25
	2	3	2	4	1			177	162	164
230	2	0	4	4	3	20	0.000010	167	158	155	0.80	12.50
	2	1	3	1	0			160	154	169

Vehicle Control: DMSO              CE: Cloning Efficiency        MF: Mutant Frequency                                        

* calculated from the mean values of the replicates of each group and rounded off to two decimal places

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that the test item, Savinyl-Feuerrot 3GLS (Savinyl Fire Red 3GLS) does not have the potential to induce gene mutation in CHO-K1 cells at the tested concentrations and under the conditions of testing employed.

Executive summary:

The genotoxic potential of the test item Savinyl-Feuerrot 3GLS (Savinyl Fire Red 3GLS) to induce gene mutation in mammalian cells was evaluated using Chinese Hamster ovary (CHO) cells.

 

The study consisted of a preliminary cytotoxicity test and a definitive gene mutation test. The gene mutation test comprised of two independent experiments, one each in the presence and absence of metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).

 

Savinyl-Feuerrot 3GLS (Savinyl Fire Red 3GLS) was soluble in Dimethyl sulphoxide (DMSO) at 200 mg/mL.

 

In a preliminary cytotoxicity test for the selection of test concentrations for the gene mutation assay, the test item showed evidence of significant cell growth inhibition as Relative Survival between 128 and 320 µg/mL both in the presence and absence of metabolic activation. There was no colony formation at 800 and 2000 µg/mL both in the presence and absence of metabolic activation, due to test item toxicity. The test item precipitated in the test medium at and above 800 µg/mL, but did not show any appreciable change in the pH and osmolality of test medium. Based on these observations a maximum of 230 µg/mL was tested in the gene mutation assay. 

 

In the gene mutation test, CHO-K1 cells were exposed to the test item in duplicate at concentrations of 4, 15, 58 and 230 µg/mL of the medium for 3 hours in the presence (Experiment 1) and absence (Experiment 2) of metabolic activation. In a similar way, a concurrent vehicle control (DMSO) and a positive control, 3-methylcholanthrene (Experiment 1) was also tested in duplicate.

 

There was no evidence of induction of gene mutations in any of the test item treated cultures either in the presence or absence of metabolic activation. In each of these experiments, the respective positive controls produced a statistically significant increase in the frequencies of mutants, under identical conditions.

 

The results of the forward gene mutation test at thehprtlocus with Savinyl-Feuerrot 3GLS (Savinyl Fire Red 3GLS) indicated that the test item was non-mutagenic under the conditions of this study.