A mixture of: sodium/potassium 7-[[[3-[[4-((2-hydroxy-naphthyl)azo)phenyl]azo]phenyl]sulfonyl]amino]-naphthalene-1,3-disulfonate

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:

16 October 2013 to 04 December 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Details on test material:

Name: FAT 45155/E TE
Batch No.: BS-DUW 1120/004-01
Physical State at RT: solid (powder)
Colour: red in fine grained form; greenish black in coarse-grained form
Density: 1.74 (relative)
Active components: 74.4%
Expiry Date: 15 August 2018
Storage Conditions: at room temperature

Specific details on test material used for the study:

Name: FAT 45155/E TE
Batch No.: BS-DUW 1120/004-01
Physical State at RT: solid (powder)
Colour: red in fine grained form; greenish black in coarse- grained form
Density: 1.74 (relative)
Active components: 74.4 %
Expiry Date: 15 August 2018
Storage Conditions: at room temperature

Method

Target gene:

hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

-Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

Pre-experiment for experiment I (with and without metabolic activation):
25, 50, 100, 250, 500, 1000, 1750, 2500, 3750, 5000 µg/mL
Pre-experiment for experiment II (only without metabolic activation, 20 h long-term exposure assay):
25, 50, 100, 250, 500, 1000, 1750, 2500, 3750, 5000 µg/mL
Experiment I
without metabolic activation: 7.4, 18.6, 37.2, 74.4, 186.0, 372.0, 446.4 and 520.8 µg/mL
and with metabolic activation: 25, 50, 100, 250, 500, 1000, 1750, 2500, 3750 and 5000 µg/mL
Experiment II
without metabolic activation: 50, 100, 200, 400, 600, 800, 1000, 1200, 1800 and 2000 µg/mL
and with metabolic activation: 35, 70, 150, 300, 600, 800, 1000, 4000 and 5000 µg/mL

Vehicle / solvent:

Vehicle (Solvent) used: cell culture medium (MEM + 0 % FBS 4h treatment; MEM + 10 % FBS 20h treatment).

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation; 300 µg/mL

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation; 0.8 and 1.0 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: dissolved in medium
DURATION: 4 h (short-term exposure), 20 h (long-term exposure)
Expression time (cells in growth medium): 5 days
Selection time (if incubation with selection agent): about one week

SELECTION AGENT (mutation assay): rate experiments (I+II) with single exposure; 5 individual flasks were seeded and evaluated
NUMBER OF CELLS EVALUATED: 400000 cells per flask
DETERMINATION OF CYTOTOXICITY: Method: relative growth

Cells:
V79 cells in vitro have been widely used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens. These cells are characterized by their high proliferation rate (12 - 14 h doubling time of the BSL BIOSERVICE stock cultures) and their high cloning efficiency of untreated cells, usually more than 50 %. These facts are necessary for the appropriate performance of the study. The V79 cells (ATCC, CCL-93) were stored over liquid nitrogen (vapour phase) in the cell bank of BSL BIOSERVICE. This allows the repeated use of the same cell culture batch in experiments. Each cell batch was routinely checked for mycoplasma infections (PCR). Thawed stock cultures were maintained in plastic culture flasks in minimal essential medium (MEM). For purifying the cell population of pre-existing HPRT- mutants cells were exposed to HAT medium containing 100 µM hypoxanthine, 0.4 µM aminopterin, 16 µM thymidine and 10.0 µM glycine for several cell doublings (2-3 days).

Evaluation criteria:

A test is considered to be negative if there is no biologically relevant increase in the number of mutants.
There are several criteria for determining a positive result:
-a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations;
-a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of
the mutant frequency is not observed;
-if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I without S9: ≥186.0 μg/mL and at 7.4 µg/mL; experiment I with S9: ≥1750 μg/mL and at 500 µg/mL; Experiment II without S9: ≥600 μg/mL; Experiment II with S9: ≥300 μg/mL

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

Precipitation:

No precipitation of the test item was noted in any of the experiments.

 

Toxicity:

A biologically relevant growth inhibition (reduction of relative growth below 70 %) was observed after the treatment with the test item in experiment I and II with and without metabolic activation.

In experiment I without metabolic activation the relative growth was 11.2 % for the highest concentration (520.8 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 5000 µg/mL with a relative growth of 21.6 %. In experiment II without metabolic activation the relative growth was 10.2 % for the highest concentration (2000 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 5000 µg/mL with a relative growth of 19.2 %.

 

Mutagenicity:

In experiment I without metabolic activation all mutant values of the negative controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-43 mutants per 10⁶ cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls. Mutation frequencies with the negative control were found to be 11.91 and 16.72 mutants/10⁶ cells and in the range of 6.41 to 33.33 mutants/10⁶ cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 2.33 was found at a concentration of 74.4 µg/mL with a relative growth of 76.2 %. With metabolic activation all mutant values of the negative controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-44 mutants per 10⁶ cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls. Mutation frequencies with the negative control were found to be 37.99 and 24.93 mutants/10⁶ cells and in the range of 13.85 to 43.30 mutants/10⁶ cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 1.38 was found at a concentration of 1750 µg/mL with a relative growth of 34.3%. In experiment II without metabolic activation most mutant values of the negative controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-43 mutants per 10⁶ cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls. Mutation frequencies with the negative control were found to be 22.63 and 25.81 mutants/106 cells and in the range of 15.05 to 58.90 mutants/106 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 2.43 was found at a concentration of 100 µg/mL with a relative growth of 72.8 %. In experiment II with metabolic activation most mutant values of the negative controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-44 mutants per 10⁶ cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls. Mutation frequencies with the negative control were found to be 33.23 and 20.60 mutants/10⁶ cells and in the range of 7.50 to 63.07 mutants/10⁶ cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 2.34 was found at a concentration of 600 µg/mL with a relative growth of 19.3 %. DMBA (0.8 and 1.0 µg/mL) and EMS (300 µg/mL) were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

Experiment I -Toxicity, without metabolic activation

Dose Group	Concen-tration[µg/mL]	Cell Density[cells/mL]^a	Relative Growth[%]^a	Number of cells per flask^b			Cloning Efficiency[%]
				I	II	mean
NC1	0	891000	100.0	143	176	160	80
NC2		889000		141	146	144	72
1	7.4	512000	57.5	143	144	144	72
2	18.6	641000	72.0	141	151	146	73
3	37.2	671000	75.4	152	174	163	82
4	74.4	678000	76.2	171	177	174	87
5	186.0	441000	49.6	155	162	159	79
6	372.0	194000	21.8	148	157	153	76
7	446.4	127000	14.3	142	150	146	73
8	520.8	100000	11.2	147	165	156	78
EMS	300	793000	89.1	135	137	136	68

NC: negative control/medium control

a: cell density and relative growth at 1^st subcultivation 

b: mean value of cells per flask/200

EMS: Ethyl methane sulfonate[300µg/ml]

Experiment I– Mutagenicity, without metabolic activation

 

Dose Group	Concen-tration[µg/mL]	Number of mutant colonies per flask^a					Mean	SD	Mutant colonies per 10^6cells^b	Mutation Factor
		I	II	III	IV	V
NC1	0	4	4	2	5	4	3.8	0.98	11.91
NC2		3	3	5	6	7	4.8	1.60	16.72
1	7.4	6	7	9	10	11	8.6	1.85	29.97	2.09
2	18.6	4	5	5	5	7	5.2	0.98	17.81	1.24
3	37.2	1	2	4	5	6	3.6	1.85	11.04	0.77
4	74.4	10	9	13	12	14	11.6	1.85	33.33	2.33
5	186.0	2	3	3	4	4	3.2	0.75	10.09	0.71
6	372.0	3	3	4	5	6	4.2	1.17	13.77	0.96
7	446.4	9	9	10	10	10	9.6	0.49	32.88	2.30
8	520.8	1	1	2	3	3	2.0	0.89	6.41	0.45
EMS	300	80	86	89	96	109	92.0	9.94	338.24	23.62

NC: negative control/medium control

a:        number of mutant colonies in flask I toV

b:        mean mutant colonies x 10⁶/ (400000 x Cloning Efficiency/100)

EMS:   Ethylmethanesulfonate [300 µg/ml]

Experiment I- Toxicity, with metabolic activation

Dose Group	Concen-tration[µg/ml]	Cell Density[cells/ml]^a	Relative Growth[%]^a	Number of cells per flask^b			Cloning Efficiency[%]
				I	II	mean
NC1	0	1230000	100.8	189	190	190	95
NC2		1210000	99.2	149	188	169	84
1	25	1270000	104.1	207	220	214	107
2	50	1180000	96.7	181	208	195	97
3	100	1110000	91.0	218	193	206	103
4	250	1180000	96.7	209	216	213	106
5	500	632000	51.8	214	177	196	98
6	1000	855000	70.1	209	215	212	106
7	1750	418000	34.3	193	195	194	97
8	2500	258000	21.1	185	198	192	96
9	3750	198000	16.2	187	197	192	96
10	5000	263000	21.6	201	189	195	98
DMBA	0.8	894000	73.3	157	162	160	80
DMBA	1.0	881000	72.2	162	182	172	86

NC:     negative control/medium control

a:        cell density and relative growth at 1^st subcultivation

 b:        mean value of cells per flask/200

DMBA: 7,12-Dimethylbenz(a)anthracene[0.8 and 1.0 µg/mL]

Experiment I– Mutagenicity, with metabolic activation

Dose Group	Concen-tration[µg/ml]	Number of mutant colonies per flask^a					Mean	SD	Mutant colonies per 10^6cells^b	Mutation Factor
		I	II	III	IV	V
NC1	0	17	20	10	14	11	14.4	3.72	37.99
NC2		5	11	11	6	9	8.4	2.50	24.93
1	25	13	15	15	16	25	16.8	4.21	39.34	1.25
2	50	11	13	14	17	18	14.6	2.58	37.53	1.19
3	100	10	12	13	14	16	13.0	2.00	31.63	1.01
4	250	8	8	8	8	9	8.2	0.40	19.29	0.61
5	500	7	7	9	9	13	9.0	2.19	23.02	0.73
6	1000	11	14	14	15	23	15.4	4.03	36.32	1.15
7	1750	14	21	15	15	19	16.8	2.71	43.30	1.38
8	2500	6	7	8	9	12	8.4	2.06	21.93	0.70
9	3750	16	16	16	17	18	16.6	0.80	43.23	1.37
10	5000	4	5	5	6	7	5.4	1.02	13.85	0.44
DMBA	0.8	40	28	27	34	34	32.6	4.72	102.19	3.25
DMBA	1.0	37	38	32	32	33	34.4	2.58	100.00	3.18

NC:     negative control/medium control

a:        number of mutant colonies in flask I to V

b:        mean mutant colonies x 10⁶/ (400000 x Cloning Efficiency/100)

DMBA: 7,12-Dimethylbenz(a)anthracene [0.8 and 1.0µg/mL]

Experiment II -Toxicity, without metabolic activation

Dose Group	Concen-tration [µg/mL]	Cell Density [cells/mL]^a	Relative Growth [%]^a	Number of cells per flask^b			Cloning Efficiency [%]
				I	II	mean
NC1	0	1890000	100.0	196	184	190	95
NC2		1820000		199	204	202	101
3	50	1510000	81.4	158	161	160	80
4	100	1350000	72.8	152	157	155	77
5	200	1830000	98.7	169	169	169	85
6	400	1310000	70.6	174	195	185	92
7	600	1060000	57.1	190	196	193	97
8	800	1020000	55.0	163	175	169	85
9	1000	805000	43.4	200	174	187	94
10	1200	574000	30.9	176	153	165	82
13	1800	142000	7.7	191	173	182	91
14	2000	189000	10.2	150	136	143	72
EMS	300	1490000	80.3	126	164	145	73

NC:negative control/medium control

a: cell density and relative growth at 1^st subcultivation

b: mean value of cells per flask /200

EMS: Ethylmethanesulfonate[300µg/ml]

Experiment II–Mutagenicity, without metabolic activation

Dose Group	Concen-tration [µg/mL]	Number of mutant colonies per flask^a					Mean	SD	Mutant colonies per 10^6cells^b	Mutation Factor
		I	II	III	IV	V
NC1	0	5	6	8	11	13	8.6	3.01	22.63
NC2		6	8	10	11	17	10.4	3.72	25.81
3	50	2	3	4	7	8	4.8	2.32	15.05	0.62
4	100	12	16	18	22	23	18.2	4.02	58.90	2.43
5	200	10	11	11	11	15	11.6	1.74	34.32	1.42
6	400	5	12	13	14	15	11.8	3.54	31.98	1.32
7	600	3	4	4	6	8	5.0	1.79	12.95	0.53
8	800	5	6	6	8	8	6.6	1.20	19.53	0.81
9	1000	4	6	8	9	11	7.6	2.42	20.32	0.84
10	1200	6	7	9	13	14	9.8	3.19	29.79	1.23
13	1800	6	7	10	11	15	9.8	3.19	26.92	1.11
14	2000	8	9	10	10	12	9.8	1.33	34.27	1.41
EMS	300	228	200	217	184	237	213.2	19.11	735.17	30.36

NC: negative control/medium control

a: number of mutant colonies in flask I to V

b: mean mutant colonies x 10⁶/ (400000 x Cloning Efficiency/100)

EMS: Ethylmethanesulfonate [300µg/ml]

Experiment II-Toxicity, with metabolic activation

Dose Group	Concen-tration [µg/mL]	Cell Density [cells/mL]^a	Relative Growth [%]^a	Number of cells per flask^b			Cloning Efficiency [%]
				I	II	mean
NC1	0	995000	100.0	157	168	163	81
NC2		902000		192	177	185	92
4	35	869000	91.6	186	179	183	91
5	70	961000	101.3	178	189	184	92
6	150	735000	77.5	182	186	184	92
7	300	510000	53.8	163	174	169	84
8	600	183000	19.3	191	180	186	93
9	800	170000	17.9	169	171	170	85
10	1000	123000	13.0	180	181	181	90
13	4000	95100	10.0	150	170	160	80
14	5000	182000	19.2	194	197	196	98
DMBA	0.8	614000	64.7	167	170	169	84
DMBA	1.0	612000	64.5	175	189	182	91

NC: negative control/medium control

a: cell density and relative growth at 1^st subcultivation

b: mean value of cells per flask/200

DMBA: 7,12-Dimethylbenz(a)anthracene[0.8and1.0µg/mL]

Experiment II–Mutagenicity, with metabolic activation

Dose Group	Concen-tration [µg/mL]	Number of mutant colonies per flask^a					Mean	SD	Mutant colonies per 10^6cells^b	Mutation Factor
		I	II	III	IV	V
NC1	0	9	11	11	11	12	10.8	0.98	33.23
NC2		5	7	7	8	11	7.6	1.96	20.60
4	35	3	4	4	10	11	6.4	3.38	17.53	0.65
5	70	7	9	9	10	14	9.8	2.32	26.70	0.99
6	150	7	9	9	10	19	10.8	4.21	29.35	1.09
7	300	6	7	8	9	13	8.6	2.42	25.52	0.95
8	600	15	23	23	25	31	23.4	5.12	63.07	2.34
9	800	4	4	4	5	6	4.6	0.80	13.53	0.50
10	1000	12	15	16	18	20	16.2	2.71	44.88	1.67
13	4000	0	2	3	3	4	2.4	1.36	7.50	0.28
14	5000	6	6	7	9	10	7.6	1.62	19.44	0.72
DMBA	0.8	109	121	124	131	133	123.6	8.52	366.77	13.63
DMBA	1.0	132	133	134	139	147	137.0	5.55	376.37	13.98

NC: negative control/medium control

a: number of mutant colonies in flask I to V

b: mean mutant colonies x 10⁶/ (400000 x Cloning Efficiency/100)

DMBA: 7,12-Dimethylbenz(a)anthracene[0.8 and 1.0µg/mL]

Applicant's summary and conclusion

Conclusions:

FAT 45155/E is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

In a GLP compliant study conducted according to OECD guideline 476 and EU method B.17, FAT 45155/E was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster. The selection of the concentrations was based on data from the pre-experiments. Experiment I with and without metabolic activation and experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as 20 h long time exposure assay. The test item was investigated at the following concentrations:

Experiment I

without metabolic activation: 7.4, 18.6, 37.2, 74.4, 186.0, 372.0, 446.4 and 520.8 µg/mL and with metabolic activation: 25, 50, 100, 250, 500, 1000, 1750, 2500, 3750 and 5000 µg/mL

Experiment II without metabolic activation: 50, 100, 200, 400, 600, 800, 1000, 1200, 1800 and 2000 µg/mL and with metabolic activation: 35, 70, 150, 300, 600, 800, 1000, 4000 and 5000 µg/mL

No precipitation of the test item was noted in the experiments. Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I without metabolic activation the relative growth was 11.2 % for the highest concentration (520.8 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 5000 µg/mL with a relative growth of 21.6 %. In experiment II without metabolic activation the relative growth was 10.2 % for the highest concentration (2000 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 5000 µg/mL with a relative growth of 19.2 %. In both experiments no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose-response relationship was observed. DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. In conclusion, FAT 45155/E is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.