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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on negative outcomes in the bacterial reverse mutation assay, chromosomal aberration study and in the mammalian cell gene mutationa assay, the substance is considered as non genotoxic.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

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

Target gene:

hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)

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).

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

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.

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

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]

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 June 1992 to 17 December 1992

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)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5375 (In Vitro Mammalian Chromosome Aberration)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: MITI Japan Notification No. 303 (1987 / Lit. 10)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Test article: FAT 45155/B
Trade name: ERIONYL ROT HT 3728
Batch No.: HT 3728/TV 1
Purity: 60 - 70 %
Physical properties: powder; dark red
Stability of the test material in the vehicle: stable
Expiration date: May 15, 1997

Target gene:

ATCC (American Type Culture Collection) CCL 61 (ovary, Chinese hamster)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Cell line: ATCC (American Type Culture Collection) CCL 61 (ovary, Chinese hamster)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat-liver post mitochondrial supernatant (S9 fraction)

Test concentrations with justification for top dose:

Experiments without metabolic activation:
- 18 hours treatment time: 39.06, 78.13 and 156.25 μg/ml
- 42 hours treatment time: 78.13, 156.25 and 312.5 μg/ml

Experiments with metabolic activation:
-3 hours treatment followed by 15 hours recovery period: 78.13, 156.25 and 312.5 μg/ml
-3 hours treatment followed by 39 hours recovery period: 156.25, 312.5 and 625 μg/ml

Vehicle / solvent:

Bidistilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

other: distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Preparation of the metabolic activation mixture:
Rat-liver post mitochondrial supernatant (S9 fraction) was prepared in advance from male RAI rats; reared at the Animal Farm of CIBA-GEIGY, Sisseln, Switzerland. The animals (150-250 g) were treated with Aroclor 1254 5 days prior to sacrifice. The livers were homogenized with 3 volumes of 150 mM KCl. The homogenate was centrifuged at 9000x g for 15 minutes and the resulting supernatant (S9 fraction) was stored at approximately -80 °C for no longer than one year. S9 fraction was thawed immediately before use, mixed with NADP and isocitric acid and added to culture medium to give the following final concentrations:
Rat liver S9 fraction 15 μl/ml (1.5 %)
NADP 3.14 μmol/ml
Isocitric acid (trisodium salt) 15.3 μmol/ml

Evaluation criteria:

Under the standard conditions of our laboratories, the test substance is generally considered to be active in the Chinese Hamster cells if the following conditions are met:
- The percentage of metaphases containing specific aberrations in a treatment group is higher than 6.0 (based on historical negative control range) and differs statistically significant from the respective value of the negative control.
- A concentration-related response should be demonstrable.

Statistics:

No data

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 156.25 μg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No data

Remarks on result:

other: all strains/cell types tested

Toxicity test

A first series of experiments have been performed using concentrations up to 5000 µg/ml.These experiments revealed strong cytotoxicity at concentrations of 156.25 µg/ml and higher in the absence of metabolic activation (18 hours) and at 625 µg/ml and higher in the presence of metabolic activation (3 hours/15 hours). No cells survived the treatment at these concentrations (data not shown on tables). Therefore, for the next series of experiments concentrations of 156.25 µg/ml (without activation) and 312.5 µg/ml (with activation) were selected as the highest.

The highest concentration of 156.25 µg/ml selected for analysis in the first experiment of the original study (without metabolic activation, 18 hours treatment) caused 16.7 % suppression of mitotic activity. The highest concentration of 312.5 µg/ml selected for analysis in the second experiment of the original study (with metabolic activation, 3 hours treatment/15 hours recovery) caused 20.0 % suppression of mitotic activity. In the 42 hours experiments of the confirmatory study concentration ranges were used up to 312.5 µg/ml (without metabolic activation) and up to 625 µg/ml (with metabolic activation). In the experiment without metabolic activation, concentrations up 312.5 µg/ml could be evaluated. The highest concentration caused 6.2 % suppression of mitotic activity. In the experiment performed with metabolic activation (3 hours treatment/39 hours recovery) concentration up to 625 µg/ml could be evaluated. The highest concentration caused 42.9 % suppression of mitotic activity.

 

Original mutagenicity study

In the experiment performed without metabolic activation (experiment 1, 18 hours treatment), 0 % of metaphases with specific chromosomal aberrations were detected in the negative control. At the concentrations of 39.06 µg/ml, 78.13 µg/ml and 156.25 Mg/ml 0 %, 1.0 % and 1.0 % of cells with specific chromosomal aberrations were found. In the experiment performed with metabolic activation (experiment 2, 3 hours treatment/15 hours recovery), 0 % of metaphases with specific chromosomal aberrations were seen in the negative control. At the concentrations of 78.13 µg/ml,156.25 µg/ml and 312.5 Mg/ml the respective values were 0 %, 1.0 % and 1.0 %. In both original experiments slight, statistically significant differences were obtained with the two higher concentrations when compared with their respective negative controls. These significances may be explained by the 0 % values obtained with the negative controls of these two experiments whereby just a small difference results in a significant response. However, since a frequency of 1 % metaphases with aberrations, as found for the treated groups, is well within the historical negative control range and far below the critical level of 6 % required for a positive response, these effects are considered to be of no biological relevance.

 

Confirmatory mutagenicity study

In the experiment performed without metabolic activation after 18 hours treatment (experiment 1), 0 % of metaphases with specific chromosomal aberrations were detected in the negative control. At the concentrations of 39.06 µg/ml/ 78.13 µg/ml and 156.25 µg/ml 0.5 %, 0.5 % and 0 % of cells with specific chromosomal aberrations were registered. In the experiment performed with metabolic activation after 3 hours treatment/15 hours recovery (experiment 2;), 0 % of metaphases with specific chromosomal aberrations were seen in the negative control. At the concentrations of 78.13 µg/ml, 156.25 Mg/ml and 312.5 µg/ml 0.5 %, 0.5 % and 0.5 % of cells showed specific chromosomal aberrations. In the experiment performed without metabolic activation after 42 hours treatment (experiment 3), 0 % of metaphases with specific chromosomal aberrations were detected in the negative control cultures. At the concentrations of 78.13 µg/ml, 156.2 5 µg/ml and 312.5 µg/ml the corresponding values were 1.5 %, 1.0 % and 0 %. In the experiment performed with metabolic activation after 3 hours treatment/39 hours recovery (experiment 4), 1.0 % of metaphases with specific chromosomal aberrations were registered in the negative control cultures. At the concentrations of 156.25 µg/ml, 312.5 µg/ml and 625 µg/ml 0 %, 0.5 % and 1.0 % of cells with specific chromosomal aberrations were found. None of these values showed a statistically significant difference when compared with their respective negative control. Unspecific chromosomal aberrations in the form of chromatid gaps found in all experiments were within the frequency generally observed.

 

Positive controls

The treatment of the cultures with mitomycin-C, 0.2 µg/ml and cyclophosphamide, 20.0 µg/ml, respectively, was followed by a high incidence of specific chromosomal aberrations in the experiments one and two of the original study (62.0 % and 46.0 %) and in the experiments one and two of the confirmatory study (28.0 % and 18.0 %).

Conclusions:

FAT 45155/B considered as non-clastogenic in Chinese hamster ovary cells.

Executive summary:

In a GLP-compliant study performed according to OECD guideline 473 and EU method EU B.10, FAT 45155/B was investigated for clastogenic (chromosome-damaging) effects on Chinese hamster ovary cells in vitro with and without extrinsic metabolic activation (S9) . The compound was dissolved in bi-distilled water and tested at each of the following conditions:

 

Experiments without metabolic activation:

- 18 hours treatment time:

original experiment: 39.06, 78.13 and 156.25 µg/ml

confirmatory experiment: 39.06, 78.13 and 156.25 µg/ml

- 42 hours treatment time: 78.13, 156.25 and 312.5 µg/ml

Final concentrations greater than 156.25 µg/ml (after 18 hours) or 312.5 µg/ml (after 42 hours) of culture medium could not be scored due to cytotoxicity. Mitomycin C (0.2 µg/ml) was used as a positive control in the 18 hours experiments.

 

Experiments with metabolic activation:

- 3 hours treatment followed by 15 hours recovery period:

original experiment: 78.13, 156.25 and 312.5 µg/ml

confirmatory experiment: 78.13, 156.25 and 312.5 µg/ml

- 3 hours treatment followed by 39 hours recovery period: 156.25, 312.5 and 625 µg/ml

Final concentrations greater than 312.5 µg/ml (after 18 hours) or 625 µg/ml (after 42 hours) of culture medium could not be scored due to cytotoxicity. Cyclophosphamide (20.0 µg/ml) was used as a positive control in the 3 hours/15 hours experiments. In addition, DNA distribution of cultures treated under the above described conditions (18 hours only) was determined by flow cytometry. These measurements allow to analyse the influence of the test substance on the cell cycle of CHO cells. In both the experiments performed without and with metabolic activation no significant increase in the number of metaphases containing specific chromosomal aberrations was observed. The incidence of aberrant cells was within the historical control range at all doses assessed.

Flow cytometry experiments did reveal evidence for very weak cell cycle disturbing activities of the test substance at the highest concentration in the presence of metabolic activation. However, the effects are considered to be of no relevance for the chromosome aberration assay. It is concluded that under the given experimental conditions no evidence of clastogenic effects was obtained in Chinese hamster ovary cells in vitro treated with FAT 45155/B.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 June 1992 to 15 December 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: MHW Japan 1984

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: EPA U.S.A 1987

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Test article: FAT 45155/B
Batch No.: HT 3728/TV 1
Purity: 60 - 70 %
Storage conditions: room temperature; keep dark
Stability of the test material in the vehicle: stable in bidistilled water
Expiration date: May 15, 1997

Target gene:

salmonella typhimurim histidine(his) reversion system

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat-liver microsomal fraction S9

Test concentrations with justification for top dose:

Range finding test: 20.5761, 61.7284, 185.1852, 555.5556, 1666.6667 and 5000.0000 μg/plate
Mutagenicity test: 61.7284, 185.1852, 555.5556, 1666.6667 and 5000.0000 μg/plate

Vehicle / solvent:

bidistilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Experiment without microsomal activation: TA 100 and TA 1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Experiment without microsomal activation: TA 98 and TA 1538

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Experiment without microsomal activation: TA1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Experiment without microsomal activation: WP2uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Experiment with microsomal activation: TA 100, TA 98, TA 1537, TA 1538 (2.5 µg/plate) and WP2uvrA (50 µg/plate)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Experiment with microsomal activation: TA 1535

Details on test system and experimental conditions:

Preparation of the bacterial cultures:
Inoculates from frozen master copies were set up monthly. They were grown in liquid NB-medium overnight and then plated on NB-agar. After incubation, single colonies were taken from the plates, grown overnight in liquid NB-medium and then used for the experiment.

Preliminary Toxicity/Range-Finding test:
A toxicity test was carried out with strain TA100 and E.COLI wp2uvrA without and with microsomal activation at six concentrations of the test substance and one negative control according to the standard operating procedures of genetic toxicology. The plates were inverted and incubated for about 48 hours at 37 ±1.5 °C in darkness.

Mutagenicity test:
The mutagenicity test was performed with strains TA 100, TA1535, TA 98, TA 1537 and E. coli WP2uvrA without and with microsomal activation according to standard operating procedures of genetic toxicology. The plates were inverted and incubated for about 48 hours at 37 ±1.5 °C in darkness. They were evaluated by counting the colonies and determine the background lawn.

Evaluation criteria:

A doubling of the spontaneous reversion rate and a dose-effect relationship.

Statistics:

In deviation to the OECD guideline a statistical analysis was not perform. No appropriate statistical method is available.

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 1538, TA100, TA 98 and E.coli

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Conclusions:

The test substance and its metobolites did not induce gene mutations in the strains of S. typhimurium and E. coli.

Executive summary:

A bacterial reverse mutation test with FAT 45155/B was carried out according to OECD guideline 471 and in compliance with GLP using in histidine-requiring strains of Salmonella typhimurium and a tryptophan-requiring strain of Escherichia coli. The concentration range of FAT 45155/B to be tested in the mutagenicity test was determined in a preliminary toxicity test. Thus, FAT 45155/B was tested for mutagenic effects without and with metabolic activation at five concentrations in the range of 61.7 to 5000 ug/plate. In order to confirm the results, the experiments were repeated in an independent experiment with the same concentrations. In a toxicity test/range finding test, no signs of toxicity of FAT 45155/B on the growth of the bacteria were observed up to the concentration of 5000 ug/ plate. While in the original mutagenicity test experiment performed without and with metabolic activation, none of the tested concentrations of FAT 45155/B led to an increase in the incidence of histidine- or tryptophanprototrophic mutants by comparison with the negative control. In the confirmatory experiment performed without and with metabolic activation, again, the tested concentrations of FAT 45155/B did not lead to an increase in the incidence of histidine- or tryptophan prototrophic mutants by comparison with the negative control. Based on the results of these experiments and on standard evaluation criteria, it is concluded that FAT 45155/B and its metabolites did not induce gene mutations in the strains of S. typhimurium and E. coli used.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Two Ames tests, one chromosome aberration test and one HPRT mutation assay were performed according to OECD guidelines with GLP.

A key Ames study was performed to define the mutagenicity of the test substance with S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 with and without metabolic activation based on OECD TG 471. There was no significantly difference for revertant between the negative controls and test groups at concentrations of 33.3; 100.0; 333.3; 1000.0; 2500.0 and 5000.0 μg/plate. It can be concluded that test substance cause no mutagenicity for S. typhimurium strains at all test concentrations. The other Ames test was performed to define the mutagenicity of the test substance with S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, TA 100 and E.coli with and without metabolic activation based on OECD TG 471. There was no significantly difference for revertant between the negative controls and test groups at concentrations of 61.7284, 185.1852, 555.5556, 1666.6667 and 5000.0000 μg/plate. It can be concluded that test substance caused no mutagenicity for S. typhimurium strains and E.coli at all test concentrations.

 

The chromosomal aberration test (Dr. Th. Hertner, 1992) was performed according to OECD TG 473 to investigated for clastogenic (chromosome-damaging) effects on Chinese hamster ovary cells in vitro with and without extrinsic metabolic activation (S9). 39.06, 78.13 and 156.25 μg/ml were applied in the test without S9 and 78.13, 156.25 and 312.5 μg/ml were applied in the test with S9. In both the experiments performed without and with metabolic activation no significant increase in the number of metaphases containing specific chromosomal aberrations was observed. The incidence of aberrant cells was within the historical control range at all doses assessed. Therefore, it can be concluded that no clastogenic effects was observed in chinese hamster ovary cells in vitro treated with the test substance under the test condition.

 

In a GLP-compliant mammalian cell gene mutation assay, tested according to OECD guideline 476, Chinese hamster V79 cells were exposed to the test substance with and without metabolic activation and the potential to induce mutations at the HPRT locus was assessed. 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 4h short-term exposure assay. Experiment II without metabolic activation was performed as 20h long time exposure assay. The following concentrations were used. Experiment I without metabolic activation: 7.4, 18.6, 37.2, 74.4, 186.0, 372.0, 446.4, and 520.8 µg/mL; Experiment I 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; Experiment II with metabolic activation: 35, 70, 150, 300, 600, 800, 1000, 4000, and 5000 µg/mL. No precipitation 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. The positive controls showed distinct biologically relevant effects in mutation frequency. In conclusion, the test substance is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese hamster.

 

All the test results above indicated no genotoxicity potential for the test substance.

Justification for classification or non-classification

Based on the findings of the genetic toxicity studies, the test substance does not considered to be classified according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.